1 Definitive guide to western blot Western blotting uses specific antibodies to identify proteins that have been separated based on size by gel electrophoresis. The immunoassay uses a membrane made of nitrocellulose or PVDF (polyvinylidene fluoride). The gel is placed next to the membrane and application of an electrical current induces the proteins to migrate from the gel to the membrane. The membrane can then be further processed with antibodies specific for the target of interest, and visualized using secondary antibodies and detection reagents. The following guide discusses the process for producing a western blot: sample preparation, gel electrophoresis, membrane transfer, and immunostaining steps. The guide is intended to be an educational resource to introduce the method and not a benchtop protocol. Please consult the manufacturers of your electrophoresis and transfer equipment for more detailed instructions on these steps. A. Sample preparation 1. Lysis buffers 2. Protease and phosphatase inhibitors 3. Preparation of lysate from cell culture 4. Preparation of lysate from tissues 5. Determination of protein concentration 6. Preparation of samples for loading into gels B. Electrophoresis 1. Preparation of PAGE gels 2. Positive controls 3. Molecular weight markers 4. Loading samples and running the gel 5. Use of loading controls C. Transfer of proteins and staining (Western blotting) 1. Visualization of proteins in gels 2. Transfer 3. Visualization of proteins in membranes: Ponceau Red 4. Blocking the membrane 5. Incubation with the primary antibody 6. Incubation with the secondary antibody 7. Development methods D. References A. Sample preparation 1. Lysis buffers To prepare samples for running on a gel, cells and tissues need to be lysed to release the proteins of interest. This disrupts the cell membrane and solubilizes intracellular proteins so they can migrate individually through the separating gel. There are many recipes for lysis buffers but a few will serve for most western blotting experiments. Broadly, they differ in their ability to solubilize proteins and buffer containing sodium dodecyl sulfate and other ionic detergents are considered to be the harshest and therefore most likely to yield high protein levels. Most Abcam antibodies recognize reduced and denatured protein and should be used under reducing and denaturing conditions. It is important to note though that some antibodies will only recognize a protein in its
2 native, non-denatured form and will not recognize a protein that has been extracted with a denaturing detergent (SDS, deoxycholate, and somewhat less denaturing, Triton X-100 and NP-40). The main consideration when choosing a lysis buffer is whether the chosen antibody will recognize denatured samples. When this is not the case, it will be noted on the antibody datasheet, and buffers without detergent or with relatively mild non-ionic detergents (NP-40k, Triton X-100) should be used. Protein location and lysis buffer choice Protein location Whole cell Cytoplasmic (soluble) Cytoplasmic (cytoskeletal bound) Membrane bound Nuclear Mitochondria Buffer recommended NP-40 or RIPA Tris-HCl Tris-Triton NP-40 or RIPA RIPA or use nuclear fraction protocol* RIPA or use mitochondrial fraction protocol* * Proteins that are found exclusively or predominantly in a sub-cellular location can be enriched in a lysate of the sub-cellular fraction compared to whole cell or tissue lysates. This can be useful when trying to obtain a signal for a weakly-expressed protein. For example, a nuclear protein will be enriched in a nuclear lysate as compared to a whole cell lysate. Sub-cellular fractions are also used to remove potentially cross-reactive proteins present in the unused fractions. Please consult our separate protocol for sub-cellular fractionation. Nonidet-P40 (NP-40) buffer 150 mm sodium chloride 1.0% NP-40 (Triton X-100 can be substituted for NP-40) 50 mm Tris, ph 8.0 This is a popular buffer studying proteins that are cytoplasmic or membrane-bound and is used for whole cell extracts. If there is concern that the protein on interest is not being completely extracted from insoluble material or aggregates, RIPA buffer may be more suitable, as it contains ionic detergents that more ready solubilize protein. RIPA buffer (Radio Immunoprecipitation Assay buffer) 150 mm sodium chloride 1.0% NP-40 or Triton X % sodium deoxycholate 0.1% SDS (sodium dodecyl sulphate) 50 mm Tris, ph 8.0 RIPA buffer is also useful for whole cell extracts and membrane-bound proteins, and may be preferred for extracting nuclear proteins. It will disrupt protein-protein interactions and may therefore be problematic for immunoprecipitation / pull down assays. The 10% sodium deoxycholate stock solution (5 g into 50 ml) must be protected from light. In cases where it is important to preserve protein-protein interactions or to minimize denaturation (for example, when it is known that the antibody to be used will only recognize a non-denatured epitope), a buffer without ionic detergents (e.g. SDS) and ideally without non-ionic detergents (e.g. Triton X-100) should be used. Cell lysis with detergent-free buffer is achieved by mechanical shearing, often with a Dounce homogenizer or by passing cells through a syringe tip. Often a simple Tris buffer will suffice, however buffers with detergents are required to release membrane - or cytoskeleton - bound proteins.
3 Tris-HCl buffer 20 mm Tris-HCl, ph 7.5 Tris-Triton buffer (Cytoskeletal proteins) 10 mm Tris, ph mm NaCl 1 mm EDTA 1 mm EGTA 1% Triton X % glycerol 0.1% SDS 0.5% deoxycholate All four of these buffers will keep at 4 C for several weeks or for up to a year aliquotted and stored at -20 C. 2. Protease and phosphatase inhibitors As soon as lysis occurs, proteolysis, dephosphorylation and denaturation begin. These events can be slowed down considerably if samples are kept on ice or at 4 C at all times and appropriate inhibitors are added fresh to the lysis buffer. Ready-to-use cocktails of inhibitors from various suppliers are available but you can make your own cocktail. Inhibitor Aprotinin Protease/phosphatase inhibited Trypsin, Chymotrypsin, Plasmin Final concentration in lysis buffer Stock (store at - 20 C) 2 µg/ml Dilute in water, 10 mg/ml. Do not reuse thawed aliquots. Leupeptin Lysosomal 5-10 µg/ml Dilute in water. Do not re-use once defrosted. Pepstatin A Aspartic proteases 1 µg/ml Dilute in methanol, 1 mm. PMSF EDTA EGTA Na Flouride Serine, Cysteine proteases Metalloproteases that require Mg++ and Mn++ Metalloproteases that require Ca++ Serine/Threonine phosphatases 1 mm Dilute in ethanol. You can re-use the same aliquot. 5 mm Dilute in dh 2 O, 0.5 M. Adjust to ph to mm Dilute in dh 2 O, 0.5 M. Adjust to ph to mm Dilute in water. Do not re-use once defrosted. Na Orthovanadate Tyrosine phosphatases 1 mm Dilute in water. Do not re-use once defrosted.
4 Sodium orthovanadate preparation All steps to be performed in a fume hood. 1. Prepare a 100 mm solution in double distilled water. 2. Set ph to 9.0 with HCl. 3. Boil until colorless. Minimize volume change due to evaporation by covering loosely. 4. Cool to room temperature. 5. Set ph to 9.0 again. 6. Boil again until colorless. 7. Repeat this cycle until the solution remains at ph 9.0 after boiling and cooling. 8. Bring up to the initial volume with water. 9. Store in aliquots at -20 C. Discard if samples turn yellow. 3. Preparation of lysate from cell culture 1. Place the cell culture dish in ice and wash the cells with ice-cold PBS. 2. Drain the PBS, then add ice-cold lysis buffer (1 ml per 107 cells/100 mm dish/150 cm 2 flask; 0.5 ml per 5x106 cells/60 mm dish/75 cm 2 flask). 3. Scrape adherent cells off the dish using a cold plastic cell scraper, then gently transfer the cell suspension into a pre-cooled microfuge tube. 4. Maintain constant agitation for 30 minutes at 4 C. 5. Centrifuge in a microcentrifuge at 4 C. You may have to vary the centrifugation force and time depending on the cell type; a guideline is 20 minutes at 12,000 rpm but this must be determined by the end-user (e.g. leukocytes need a very light centrifugation). 6. Gently remove the tubes from the centrifuge and place on ice, aspirate the supernatant and place in a fresh tube kept on ice, and discard the pellet. 4. Preparation of lysate from tissues 1. Dissect the tissue of interest with clean tool, on ice preferably, and as quickly as possible to prevent degradation by proteases. 2. Place the tissue in round bottom microfuge tubes of Eppendorf tubes and immerse in liquid nitrogen to snap freeze. Store samples at -80 C for later use or keep on ice for immediate homogenization. For a ~5 mg piece of tissue, add ~300 µl lysis buffer rapidly to the tube, homogenize with an electric homogenizer, rinse the blade twice with another 2x300 µl lysis buffer, then maintain constant agitation for 2 hours at 4 C (e.g. place on an orbital shaker in the fridge). Volumes of lysis buffer must be determined in relation to the amount of tissue present (protein extract should not be too diluted to avoid loss of protein and large volumes of samples to be loaded onto gels. The minimum concentration is 0.1 mg/ml, optimal concentration is 1-5 mg/ml). 3. Centrifuge for 20 minutes at 12,000 rpm at 4 C in a microcentrifuge. Gently remove the tubes from the centrifuge and place on ice, aspirate the supernatant and place in a fresh tube kept on ice; discard the pellet. The buffer (with inhibitors) should be ice-cold prior to homogenization. 5. Determination of protein concentration Perform a Bradford assay, a Lowry assay, or a BCA assay. Bovine serum albumin (BSA) is a frequently-used protein standard. Once you have determined the concentration of each sample, you can freeze them at -20 C or -80 C for later use or prepare for immunoprecipitation or for loading onto a gel.
5 6. Preparation of samples for loading into gels: denatured and native, reduces and nonreduced. A denatured, reduced samples. Antibodies typically recognize a small portion of the protein of interest (referred to as the epitope) and this domain may reside within the 3D conformation of the protein. To enable access of the antibody to this portion it is necessary to unfold the protein, i.e. denature it. To denature, use a loading buffer with the anionic denaturing detergent sodium dodecyl sulfate (SDS), and boil the mixture at C for 5 minutes. Heating at 70 C for 5-10 minutes is also acceptable and may be preferable when studying multi-pass membrane proteins. These tend to aggregate when boiled and the aggregates may not enter the gel efficiently. The standard loading buffer is called 2X Laemmli buffer, first described in Nature, 1970 Aug 15; 227 (5259): It can also be made at 4X and 6X strength to minimize dilution of the samples. The 2X is to be mixed in a 1:1 ratio with the sample. Laemmli 2X buffer 4% SDS 10% 2-mercaptoehtanol 20% glycerol 0.004% bromophenol blue M Tris HCl Check the ph and bring it to ph 6.8. When SDS is used with proteins, all of the proteins become negatively charged by their attachment to the SDS anions. SDS denatures proteins by wrapping around the polypeptide backbone. SDS binds to proteins fairly specifically in a mass ratio of 1.4:1. In doing so, SDS confers a negative charge to the polypeptide in proportion to its length i.e. the denatured polypeptides become rods of negative charge clouds with equal charge densities per unit length. In denaturing SDS-PAGE separations, therefore, migration is determined not by intrinsic electrical charge of the polypeptide, but by molecular weight. The quality of SDS is of utmost importance: old or poor quality SDS can lead to artifacts such as a smear of protein along the entire gel track. It is usually necessary to reduce disulphide bridges in proteins by using ß-mercaptoethanol or dithiothreitol (DTT). This allows proteins to adopt the randomcoil configuration necessary for separation by size. Glycerol is added to the loading buffer to increase the density of the sample to be loaded and hence maintain the sample at the bottom of the well, restricting overflow and uneven gel loading. To enable visualization of the migration of proteins it is common to include in the loading buffer a small anionic dye molecule (e.g. bromophenol blue). Since the dye is anionic and small, it will migrate the fastest of any component in the mixture to be separated and provide a migration front to monitor the separation progress. During protein sample treatment the sample should be mixed by vortexing before and after the heating step for best resolution. B Native and non-reduced samples. Alternatively, an antibody may recognize an epitope made up of non-contiguous amino acids. Although the amino acids of the epitope are separated from one another in the primary sequence, they are close to each other in the folded three-dimensional structure of the protein, and the antibody will only recognize the epitope as it exists on the the surface of the folded structure.
6 It is imperative, in these circumstances, to run a western blot in non-denaturing conditions, and this will be noted on the datasheet in the applications section. In general, a non-denaturing condition simply means leaving SDS out of the sample and migration buffers and not heating the samples. Certain antibodies only recognize protein in its non-reduced form i.e. in an oxidized form (particularly on cysteine residues) and the reducing agents ß-mercaptoethanol and DTT must be left out of the loading buffer and migration buffer (non-reducing conditions). Protein state Gel condition Loading buffer Migration buffer Reduced Reducing & With β- With SDS denatured denaturing mercaptoethanol or Reduced native Oxidized denatured Oxidized native Reducing & nondenaturing Non-reducing & denaturing Non-reducing & native DTT, with SDS With β- mercaptoethanol or DTT, no SDS No β- mercaptoethanol or DTT, with SDS No β- mercaptoethanol or DTT, no SDS No SDS With SDS No SDS Rule of thumb: Reduce and denature unless the datasheet specifies otherwise. B. Electrophoresis Electrophoresis can be one dimensional (i.e. one plane of separation) or two dimensional. One dimensional electrophoresis is used for most routine protein and nucleic acid separations. Two dimensional separation of proteins is used for finger-printing, and when properly constructed can be extremely accurate in resolving all of the proteins present within a sample. Here we will be describing techniques for one dimensional electrophoresis. We recommend Gel Electrophoresis of Proteins: A Practical Approach (3 rd Edition, B.D. Hames and D. Rickwood, The Practical Approach Series, Oxford University Press, 1998) as a reference for basic understanding of 2D electrophoresis protocols. 1. Preparation of PAGE gels When separated on a polyacrylamide gel, the procedure is abbreviated as SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis). The technique is a standard means for separating proteins according to their molecular weight. Polyacrylamide gels are formed from the polymerization of two compounds, acrylasmide and N, N- methylenebisacrylamide (Bis for short). Bis is a cross-linking agent for the gels. The polymerization is initiated by the addition of ammonium persulfate along with either DMAP or TEMED. The gels are neutral, hydrophilic, three-dimensional networks of long hydrocarbons cross-linked by methylene groups. The separation of molecules within a gel is determined by the relative size of the pores formed within the gel. The pore size of a gel is determined by two factors: the total amount of acrylamide present (designated as %T) and the amount of cross-linker (%C). As the total amount of acrylamide increases, the pore size decreases. With cross-linking, 5%C gives the smallest pore size. Any increase or decrease in %C increases the pore size. Gels are designated as percent solutions and will have two necessary parameters. The total acrylamide is given as a percentage (w/v) of the acrylamide plus the bis-acrylamide. Thus, a 7.5%T would indicate that there is a total of
7 7.5 g of acrylamide and bis per 100 ml of gel. A gel designated as 7.5%T:5%C would have a total of 7.5% (w/v) acrylamide + bis, and the bis would be 5% of the total (with pure acrylamide composing the remaining 2.5%). Gels can be purchased ready-made or produced in the laboratory (recipes can be found in laboratory handbooks). The Abcam laboratory uses gels from our Optiblot range. Either way, choose carefully the percentage of you gel as this will determine the rate of migration and degree of separation between proteins. Rule of thumb: The smaller the size of the protein of interest, the higher the percentage of mono/bis. The bigger the size of the protein of interest, the lower the percentage of mono/bis. Protein size (kda) Gel percentage (%) Acrylamide is a potent cumulative neurotoxin: wear gloves at all times. Place gels in the electrophoresis tank as instructed by the manufacturer and bathe in migration buffer. 2. Positive controls Positive control lysate is used to demonstrate that the protocol is efficient and correct and that the antibody recognizes the target protein which may not be present in the experimental samples. We strongly recommend the use of a positive control lysate when setting up a new experiment; this will give you immediate confidence in the protocol. 3. Molecular weight markers A range of molecular weight markers will enable the determination of the protein size (see below) and also to monitor the progress of an electrophoretic run. A range of MW markers are commercially available. Molecular Weight Marker (ab48854) Prism Protein Ladder ( kda) (ab115832) Prism Ultra Protein Ladder ( kda) (ab116027) Prism Ultra Protein Ladder ( kda) (ab116028) Prism Ultra Protein Ladder ( kda) (ab116029) 4. Loading samples and running the gel Use special gel loading tips or a micro-syringe to load the complete sample in a narrow well. Take care not to poke the well bottom with the tip as this will create a distorted band. Never overfill wells. This could lead to poor data if samples spill into adjacent wells, and poorly resolved bands. Load µg total protein per mini-gel well. The gels will be submerged in migration buffer which normally contains SDS, except in native gel electrophoresis.
8 A standard migration buffer (also called running bugger) for PAGE is 1X Tris-glycine: 25 mm Tris base 190 mm glycine 0.1% SDS Check the ph; it should be around 8.3. Run the gel for the recommended time as instructed by the manufacturer; this can vary from machine to machine (1 hour to overnight depending on the voltage). When the dye molecule (the migration front ) reached the bottom of the gel, the power is turned off. Proteins will slowly elute from the gel at this point, so do not store the gel; proceed immediately to transfer. 5. Loading controls Loading controls are required to check that the lanes in your gel have been evenly loaded with sample, especially when a comparison must be made between the expression levels of a protein in different samples. They are also useful to check for even transfer have not occurred, the loading control bands can be used to quantify the protein amounts in each lane. For publication-quality work, use of a loading control is absolutely essential. Loading control Sample type kda Caution Vinculin Whole cell 125 Cyclophilin B Whole cell 24 GAPDH Whole cell 35 Some physiological factors, such as hypoxia and diabetes, increase GAPDH expression in certain cell types. Cofilin Whole cell / Nuclear / 19 Membrane / Cytoskeleton Alpha tubulin Whole cell / Cytoskeleton 50 Tubulin expression may vary according to resistance to antimicrobial and antimitotic drugs (Sangrajrang S, et al, 1998, Prasad V et al, 2000). Beta tubulin Whole cell / Cytoskeleton 50 Tubulin expression may vary according to resistance to antimicrobial and antimitotic drugs (Sangrajrang S, et al, 1998, Prasad V et al, 2000). Actin Whole cell / Cytoskeleton 42 Beta actin Whole cell / Cytoskeleton 40 Not suitable for skeletal muscle samples. Changes in cell-growth conditions and interactions with extracellular matrix components may after actin protein synthesis (Farmer et al, 1983). VDAC1/Porin Mitochondrial 30 COX IV Mitochondrial 20 Many proteins run at the same 16 kda size as COX IV.
9 HSP60 Mitochondrial/Membrane 60 Lamin B1 Nuclear 66 Not suitable for samples where the nuclear envelope is removed. HDAC1 Nuclear 55 YY1 Nuclear 45 TBP Nuclear 35 Not suitable for samples where DNA is removed. PCNA Nuclear 30 cdk4 Nuclear/Membrane 34 Nak ATPase Membrane 110 Transferrin Serum 75 C. Transfer of proteins and staining 1. Visualization of proteins in gels This visualization of proteins within the SDS-PAGE gel is useful to determine if proteins have electrophoresed uniformly and evenly. Use the copper stain if you plan to transfer the separated proteins to a membrane, as the Coomassie stain is not reversible. Only use the Coomassie stain on gels post-transfer to check the efficiency of the transfer, or if you have no plans to transfer and just want to observe the results of the SDS-PAGE separation. a) Coomassie stain As soon as the power is turned off the separated protein bands will begin to diffuse (they are freely soluble in aqueous solution). To prevent diffusion of proteins treat the gel with a 40% distilled water, 10% acetic acid, and 50% methanol solution which causes almost all proteins to precipitate (become insoluble). To visualize the fixed proteins place the gel in the same mixture of water/acetic acid/methanol but with the addition of 0.25% by weight Coomassie Brilliant Blue R-259. Incubate 4 hours to overnight at room temperature on a shaker. Transfer the gel (save the dye mixture; it can be re-used many times) to a mixture of 67.5% distilled water, 7.5% acetic acid, and 25% methanol, place on shaker, and replace with fresh rinse mixture until the excess dye has been removed. The stain will not bind to the acrylamide, and it will wash out (leaving a clear gel). However, it remains strongly bound to the proteins in the gel, and these take on a deep blue color. b) Copper stain Briefly rinse freshly-electrophoresed gels in distilled water (30 seconds maximum) and then transfer to 0.3 M CuCl2 for 5 to 15 minutes. Wash the gels briefly in de-ionized water, and view them against a dark-field background. Proteins come up as clear zones in a translucent blue background. Gels may be destained completely by repeated washing in M Tris/0.25 M EDTA ph 8.0. Move the gel to a dish of transfer buffer before proceeding with transfer according to the transfer apparatus manufacturer s instructions. 2. Transfer Detailed instructions for the transfer process can be found on the websites of the manufacturers of transfer apparatus, and will vary depending on the system. The principle is the same in each case though. Just as proteins with an electrical charge (conferred by the SDS bound to them) can be induced to travel through a gel in an electrical field, so too can the proteins be transferred in an electrical field from the gel onto a sturdy support, a membrane that blots the proteins from the gel. Early methods relied on diffusion; blotting in an electrical field is now standard. Transfer can be done in wet or semi-dry conditions. Semi-dry transfer is generally faster but wet transfer is less prone to failure due to drying of the membrane and is especially recommended for proteins greater than 100
10 kda. For both kinds of transfer, the membrane is placed next to the gel. The two are sandwiched between absorbent materials, and the sandwich is clamped between solid supports to maintain tight contact between the gel and membrane. In wet transfer, the gel and membrane are sandwiched between sponge and paper (sponge/paper/gel/membrane/paper/sponge) and all are clamped tightly together after ensuring no air bubbles have formed between the gel and membrane. The sandwich is submerged in transfer buffer to which an electrical field is applied. The negatively-charged proteins travel towards the positively-charged electrode, but the membrane stops them, binds them, and prevents them from continuing on. A standard buffer for wet transfer is the same as the 1X Tris-glycine buffer used for the migration/running buffer without SDS but with the addition of methanol to a final concentration of 20%. For proteins larger than 80 kd, it is recommended that SDS is included at a final concentration of 0.1%. In semi-dry transfer, a sandwich of paper/gel/membrane/paper wetted in transfer buffer is placed directly between positive and negative electrodes (cathode and anode respectively). As for wet transfer, it is important that the membrane is closest to the positive electrode and the gel closest to the negative electrode. The proportion of Tris and glycine in the transfer buffer is not necessarily the same as for wet transfer; consult the apparatus manufacturer s protocol. A standard recipe is 48 mm Tris, 39 mm glycine, 0.04% SDS, 20% methanol. Two types of membranes are available: nitrocellulose and PVDF (positively-charged nylon). The choice is personal and both work very well. PVDF membranes require careful pre-treatment: cut the membrane to the appropriate size then soak it in methanol for 1-2 minutes. Incubate in ice cold transfer buffer for 5 minutes. The gel needs to equilibrate for 3-5 minutes in ice cold transfer buffer. Failure to do so will cause shrinking while transferring, and a distorted pattern of transfer. Note on transfer of large and small proteins The amount of SDS and methanol in the transfer buffer, protein size, and gel percentage can affect transfer efficiency. The following modifications will encourage efficient transfer. Large proteins (>100 kd). 1. For large proteins, transfer out of the gel may be very slow, just as they run slowly within the gel during separation. If blotting a large protein, be sure to run your samples in a low-concentration gel, 8% or less. These will be very fragile, so handle carefully. 2. Large proteins will tend to precipitate in the gel, hindering transfer. Adding SDS to a final concentration of 0.1% in the transfer buffer will discourage this. Methanol tends to remove SDS from proteins, so reducing the methanol percentage to 10% or less will also guard against precipitation. 3. Lowering methanol in the transfer buffer also promotes swelling of the gel, allowing large proteins to transfer more easily. 4. Methanol is only necessary if using nitrocellulose. If using PVDF, methanol can be removed from the transfer buffer altogether, and is only needed to activate the PVDF before assembling the gel/membrane sandwich. 5. Choose wet transfer overnight at 4 C instead of semi-dry transfer. Small proteins (<100 kd) 1. All proteins are hindered from binding to membranes by SDS but small proteins more so than large proteins. If your protein of interest is small, consider removing SDS from the transfer buffer. 2. Keep the methanol concentration at 20%. The following reference discusses a gel and buffer system that allows transfer of proteins as larfe as 500 kd: Bolt and Mahoney, High-efficiency blotting of proteins of diverse sizes following sodium dodecyl sulfatepolyacrylamide, gel electrophoresis. Analytical Biochemistry 247, (1997).
11 Avoid touching the membrane with your fingers; use tweezers instead. Oils and proteins on the fingers will block efficient transfer and create dirty blots. After sandwiching the gel and membrane between paper, air bubbles between the fel and membrane can be removed by rolling them out with a pipet or 15 ml tube, or by assembling the sandwich in a dish of transfer buffer to prevent formation of bubbles in the first place. Wear gloves! Make sure the paper and membrane are cut to the same size as the gel. Large overhangs may prevent a current from passing through the membrane in semi-dry transfers. Chicken antibodies tend to bind to PVDF and other nylon-based membranes, leading to high background. Switching to a nitrocellulose membrane should help reduce background staining. 3. Visualization of proteins in membranes: Ponceau Red 1. To check for success of transfer, wash the membrane in TBST (for a TBST recipe, see below). Dilute the stock Ponceau Red 1:10. The stock is made of 2% Ponceau S in 30% trichloracetic acid and 30% sulfosalicylic acid. 2. Incubate on an agitator for 5 minutes. 3. Wash extensively in water until the water is clear and the protein bands are well defined. 4. The membrane may be destained completely by repeated washing in TBST or water. When using a PVDF membrane, re-activate the membrane with methanol then wash again in TBST. TBS 10x (concentrated TBS) g Trizma HCl/ g NaCl Mix in 800 ml ultra pure water ph to 7.6 with pure HCl Top up to 1L TBST For 1 L: 100 ml of TBS 10x ml ultra pure water + 1 ml Tween20 Tween20 is very viscous and will stick to the tip of your measuring pipettes. Be sure you add the right amount of the detergent to the Tris buffer. A 10% solution is easier to dispense than undiluted Tween Blocking the membrane Blocking the membrane prevents non-specific background binding of the primary and/or secondary antibodies to the membrane (which has a high capacity for binding proteins and therefore antibodies). Two blocking solutions are traditionally used: non-fat milk or BSA (Cohn fraction V). Milk is cheaper but is not recommended for studies of phospho-proteins (milk contains casein which is a phospho-protein; it causes high background because the phosphor-specific antibody detects the casein present in the milk). Some antibodies give a stronger signal on membranes blocked with BSA as opposed to milk for unknown reasons. Check the application notes on the datasheet in case there are specific instructions on how to block the membrane. To prepare a 5% milk or BSA solution, weigh 5 g per 100 ml of Tris Buffer Saline Tween20 (TBST) buffer. Mix well and filter. Failure to filter can lead to spotting where tiny dark grains will contaminate the blot during development. Incubate for 1 hour at 4 C under agitation. Rinse for 5 seconds in TBST after the incubation.
12 5. Incubation with the primary antibody Incubation buffer: Dilute the antibody in TBST at the suggested dilution, if the datasheet does not have a recommended dilution try a range of dilutions (1:100-1:3000) and optimize the dilution according to the results. Too much antibody will result in non-specific bands. It is traditional in certain laboratories to incubate the antibody in blocking buffer, while other laboratories incubate the antibody in TBST without a blocking agent. The results are variable from antibody to antibody and you may find it makes a difference to either use no blocking agent in the antibody buffer or the same agent as the blocking buffer. If high background is not an issue, some antibodies produce a much stronger signal if diluted in buffer with low concentrations ( %) of milk or BSA, or none at all. Incubation time: The time can vary between a few hours and overnight (rarely more than 18 hours), and is dependent on the binding affinity of the antibody for the protein and the abundance of protein. We recommend a more dilute antibody and a prolonged incubation to ensure specific binding. Incubation temperature: Preferably cold. In incubating in blocking buffer overnight, it is imperative to incubate at 4 C or contamination will incur and thus destruction of the protein (especially phosphor groups). Agitation of the antibody is recommended to enable adequate homogenous covering of the membrane and prevent uneven binding. 6. Incubation with secondary antibody Wash the membrane several times in TBST while agitating, 5 minutes or more per wash, to remove residual primary antibody. Incubation buffer and dilution: Dilute the antibody in TBST at the suggested dilution. If the datasheet does not have a recommended dilution, try a range of dilutions (1:1000-1:20,000) and optimize the dilution according to the results. Too much antibody will result in non-specific bands. You may incubate the secondary antibody (and primary antibody) in blocking buffer, but a reduction in background may come at the cost of a weaker specific signal, presumably because the blocking protein hinders binding of the antibody to the target protein. Incubation time and temperature: 1-2 hours, room temperature, with agitation. Which conjugate? We recommend HRP-conjugated secondary antibodies. ALP-conjugated secondary antibodies (alkaline phosphatase) are not recommended as they are not sensitive enough. 7. Development methods Detection kits For HRP-conjugated antibodies: ECL and ECL+ (home made or commercially available) are the traditional kits used and we recommend EDL+. For the new generation detection machines such as Genegnome, use the detection kit recommended by the manufacturer of the machine. We do not recommend ECL or BCIP/NBT detection kits as they are not as sensitive. X-ray films Manual film development is traditionally used and enables the scientist to control the incubation time of the x-ray film in the developing agent and fixation agent.
13 Automated x-ray film developers are also widely used and easy to use. Remember that an over-exposed film is not suitable for analysis as determination of the relative amount of protein is not possible. Over-exposed films show totally black bands with no contrast, and/or numerous non-specific bands. Digital images The new generation of film developers are units with a camera inside an enclosure, removing the need for a darkroom. The camera detects the chemiluminescence emanating from the membrane, transforming the signal into a digital image for rapid analysis with software provided with the detection machine. A range of machines are now commercially available. At the front of the next generation are systems which do not use HRP-conjugated antibodies (i.e. chemilumenescence): for example, STORM Analyzers detect fluorescence from fluorochrome-conjugated secondary antibodies. The Odyssey Infrared Imaging System detects infrared fluorescence. D. References Harlow, Ed, and David Lane. Using antibodies. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press, B.D. Hames and D. Rickwood. Gel Electrophoresis of Proteins: A Practical Approach 3 rd Edition, The Practical approach series, Oxford University Press, Bold and Mahoney, High-efficiency blotting of proteins of diverse sizes following sodium dodecyl sulfatepolyacrylamide, gel electrophoresis. Analytical Biochemistry 257, , 1997.
WESTERN BLOTTING - A BEGINNER S GUIDE Western blotting identifies with specific antibodies proteins that have been separated from one another according to their size by gel electrophoresis. The blot is
Western Blotting: Mini-gels Materials a Protein Extraction Buffer (for callus or kernel), Solution Stock Final Volume Tris-HCl ph 80 1 M 200 mm 20 ml NaCl 4 M 100 mm 25 ml Sucrose 2 M 400 mm 20 ml EDTA
Western Blotting All steps are carried out at room temperature unless otherwise indicated. Recipes for all solutions highlighted bold are included at the end of the protocol. SDS-PAGE 1. Construct an SDS-PAGE
Western Blotting Sive Lab Protocol March 2007 Prepare samples: For zebrafish embryos: Option 1: Take live embryos and put into 1.5 ml tube with E3. Centrifuge gently for 1-2 minutes -yolk lipids will rise
WESTERN BLOTTING TIPS AND TROUBLESHOOTING GUIDE TIPS FOR SUCCESSFUL WESTERB BLOTS TROUBLESHOOTING GUIDE 1. Suboptimal protein transfer. This is the most common complaint with western blotting and could
CHRISTIAN LAB WESTERN BLOT PROTOCOL There is actually 2 parts to a western blot: A. SDS-PAGE: Separates protein by size. Smaller proteins migrate faster through the gel than larger proteins. Size separation
Running protein gels and detection of proteins 1. Protein concentration determination using the BIO RAD reagent This assay uses a colour change reaction to give a direct measurement of protein concentration.
IMMUNOPRECIPITATION (IP) PROTOCOL Immunoprecipitation is a method that enables the purification of a protein. An antibody for the protein of interest is incubated with a cell extract so that the antibody
EZ-Run Protein Gel Solution EZ-Run Protein Standards EZ-Run Gel Staining Solution Traditional SDS-Page Reagents Protein Electrophoresis protein electrophoresis Introduction Sodium dodecyl sulfate polyacrylamide
METHOD USED TO EXTRACT TOTAL MUSCLE PROTEIN FOR WESTERN BLOT USING TRIS-EDTA BUFFER* SOLUTIONS FOR SAMPLE EXTRACTION 1. Tris-EDTA Buffer, ph 8.3 1 liter 50 mm Tris 6.06 g 10 mm EDTA 3.72 g Adjust ph to
Method for Western Blotting Western Blotting ELECTROPHORESIS Prepare and run an SDS PAGE gel. Select a gel percent which will give the best resolution for the size of antigen being analyzed (if known).
Electrophoresis and Electroblotting of Proteins The purpose of the next lab exercises will be to study the relative amounts of β-actin in cells of the B-16 melanoma, liver and muscle of mice. Electrophoresis
Protocol for Western Blotting Materials Materials used on Day 3 Protease inhibitor stock: 1 μg/μl pepstatin A in DMSO 200 μm leupeptin in OG Buffer 200 mm PMSF: Freshly made. Ex) 34.8 mg PMSF in 1 ml isopropanol
Western Blot Analysis with Cell Samples Grown in Channel-µ-Slides Polyacrylamide gel electrophoresis (PAGE) and subsequent analyses are common tools in biochemistry and molecular biology. This Application
Western Blot Protocol (updated on 05/20/14) Required Solutions 10x PBS (1L) 80 g NaCl 2 g KCl 14.4 g Na 2 HPO 4 or 22 g Na 2 HPO 4 7H 2 O 2.4 g KH 2 PO 4 or 2 g KH 2 PO4 Adjust ph to 7.4 Autoclave PBST
SDS-PAGE Protocol Mutated from the SDS-PAGE protocol written by the Lord of the Flies Pouring the resolving gel 1. Clean glass plates with soap and water, then with ethanol. Assemble the glass plates and
Chromatin Immunoprecipitation (ChIP) Day 1 A) DNA shearing 1. Samples Dissect tissue (One Mouse OBs) of interest and transfer to an eppendorf containing 0.5 ml of dissecting media (on ice) or PBS but without
HIS-Select Nickel Affinity Gel Catalog Number P6611 Storage Temperature 2 8 C TECHNICAL BULLETIN Product Description HIS-Select Nickel Affinity Gel is an immobilized metalion affinity chromatography (IMAC)
Western Blotting For Protein Analysis Part 1: Laemmli Gel Electrophoresis Using Mini-PROTEAN II Electrophoresis Cell Note: Powder-free gloves should be worn throughout the entire procedure. A. Preparing
292PR 01 G-Biosciences 1-800-628-7730 1-314-991-6034 firstname.lastname@example.org A Geno Technology, Inc. (USA) brand name Classic Immunoprecipitation Utilizes Protein A/G Agarose for Antibody Binding (Cat.
PROTOCOL Western Blotting Transfer and Detection Procedure 1850 Millrace Drive, Suite 3A Eugene, Oregon 97403 02-11 DESCRIPTION Western Blotting Transfer and Detection Procedure ADDITIONAL MATERIALS REQUIRED
SDS-PAGE and western blot for low molecular weight proteins (2-20kDa) Merav Marom Shamur, Smart Assays Aim: Analysis of low molecular weight proteins by SDS-PAGE and western blot under reducing conditions.
Product Manual Pure-IP Western Blot Detection Kit Catalog Number PRB-5002 20 blots FOR RESEARCH USE ONLY Not for use in diagnostic procedures Introduction The technique of immunoprecipitation (IP) is used
A Guide to Protein Blotting SDS-PAGE SDS-PAGE stands for sodium dodecyl (lauryl) sulphate-polyacrylamide gel electrophoresis. SDS-PAGE has a number of uses, which include: establishing protein size, protein
PR110 G-Biosciences 1-800-628-7730 1-314-991-6034 email@example.com A Geno Technology, Inc. (USA) brand name Dot Blot Analysis Teacher s Guidebook (Cat. # BE 502) think proteins! think G-Biosciences
Anti-ATF6 α antibody, mouse monoclonal (1-7) 73-500 50 ug ATF6 (activating transcription factor 6) is an endoplasmic reticulum (ER) membrane-bound transcription factor activated in response to ER stress.
Technical Manual ECL Western Blotting Substrate INSTRUCTIONS FOR USE OF PRODUCTS W1001 AND W1015. PRINTED IN USA. 6/09 ECL Western Blotting Substrate All technical literature is available on the Internet
STANDARD OPERATING PROCEDURE Title: Evaluation using Western Blot SOP#: M-103 Version #: 1 Author: R. Saul Date Approved: Feb. 5, 2009 Date Modified: 1. PURPOSE The purpose of this document is to describe
Protein Detection Methods & Western Blots 1.Sodium DodecylSlufate(SDS)-PolyacrylamideGel Electrophoresis (SDS-PAGE) Visualize many proteins at once 2. SDS-PAGE combined with immunoblotting (Western) Visualize
Chromatin Immunoprecipitation A) Prepare a yeast culture (see the Galactose Induction Protocol for details). 1) Start a small culture (e.g. 2 ml) in YEPD or selective media from a single colony. 2) Spin
Background When an electrical field is applied across a solution, the movement of the charged particles (proteins) is influenced not only by the charge but also the voltage, distance between electrodes,
Western Blot SOP Protein transfer from SDS-PAGE to nitrocellulose membrane using the Trans-Blot SD cell (Western). Date: 8/16/05, 10/31/05, 2/6/06 Author: N.Oganesyan, R. Kim Edited by: R. Kim Summary:
7 Electrophoresis Objectives: A) To perform agarose gel electrophoresis of the proteins isolated in last week's experiment and B) to interpret the banding patterns produced by these proteins. Introduction:
Approaches that can be used to study expression of specific proteins Receptors and transporters Homogenate binding studies Receptor autoradiography Radiochemical Western blotting Immunohistochemistry/cytochemistry
Section XIII: In This Section Introduction 196 Buffers for in Agarose 197 Casting Agarose Gels for 198 Preparation and Loading of Protein Samples 198 Optimal Voltage and Electrophoretic Times 199 Detection
BUFFERS and MEDIAS Coomassie Blue Staining Solution 2 g Coomassie Blue 2 L Methanol or Ethanol * 1.6 L 400 ml Glacial acetic acid *If you will be microwaving the gel in staining solution for rapid staining
ProteoSilver Plus Silver Stain Kit Product Code PROT-SIL2 Store at Room Temperature TECHNICAL BULLETIN Product Description The ProteoSilver Plus Silver Stain Kit is an exceptionally sensitive, MALDI-MS
Buffer Recipes For 2DE Always use electrophoresis/molecular biology quality reagents or higher Always use 18.2 ohm water quality or higher (MilliQ or bottled) As indicated in individual buffers, some reagents
CLONTECH Innovative Tools to Accelerate Discovery Antibodies Protocol Guide PT3407-1 (PR99224) Published 14 September 1999 FOR RESEARCH USE ONLY Table of Contents I. Introduction 3 II. Materials Required
Western Blot Protocol 1. Cast Gel: Assemble minigel apparatus( Be sure no leaking) Make resolution gel (recipe) and mix Make stacking gel (recipe) diwater acrylamide/bis Add 7.5ml of res gel to plates;
Southern Blot Analysis (from Baker lab, university of Florida) DNA Prep Prepare DNA via your favorite method. You may find a protocol under Mini Yeast Genomic Prep. Restriction Digest 1.Digest DNA with
Troubleshooting Guide for Electrophoresis. ELECTROPHORESIS Protocols and Recommendations for Electrophoresis electrophoresis problem 1 Low intensity of all or some bands 2 Smeared bands 3 Atypical banding
Discontinuous native protein gel electrophoresis Michael Niepmann and Junfeng Zheng Institute of Biochemistry Friedrichstrasse 24 Faculty of Medicine, JustusLIebigUniversity 35392 Giessen, Germany In this
AES Application Focus Blotting Page 1 Western Blotting Adapted from Chapter 7, Gel Electrophoresis of Proteins, by David E. Garfin, Pages 197-268, in Essential Cell Biology, Volume 1: Cell Structure, A
Nitrotyrosine Western blot starter pack Cat no. A010-513 A convenient reagent pack containing Badrilla s high integrity nitrotyrosine monoclonal antibody (clone 2E11) and associated positive and negative
Protein immunoblotting (Western blotting) Dr. Serageldeen A. A. Sultan Lecturer of virology Dept. of Microbiology SVU, Qena, Egypt firstname.lastname@example.org Western blotting -It is an analytical technique used to
Chem 405 Biochemistry Lab I Experiment 2 Quantitation of an unknown protein solution. Introduction: The determination of protein concentration is frequently required in biochemical work. Several methods
2D gel Protocol 1. Lysis and Protein Extraction from cells Prepare cell lysates with Trizol extraction by following Kathleen Lyons s protocol: AfCS Procedure Protocol PP00000155, Version 1, 05/12/03 (Ref.1).
Plant Genomic DNA Extraction using CTAB Introduction The search for a more efficient means of extracting DNA of both higher quality and yield has lead to the development of a variety of protocols, however
Instruction Manual NuPAGE Technical Guide General information and protocols for using the NuPAGE electrophoresis system Version E October 1, 2003 IM-1001 2 Table of Contents Table of Contents...3 General
Benchtop Mitochondria Isolation Protocol Note: Specific protocols are available for the following products: MS850 Mitochondria Isolation Kit for Rodent Tissue MS851 Mitochondria Isolation Kit for Rodent
Chapter 3 Contd. Western blotting & SDS PAGE Western Blot Western blots allow investigators to determine the molecular weight of a protein and to measure relative amounts of the protein present in different
Pharmaceutical Biotechnology Recombinant DNA technology Western blotting and SDS-PAGE Recombinant DNA Technology Protein Synthesis Western Blot Western blots allow investigators to determine the molecular
Express TM PAGE Gels Technical Manual No. 0210 Update date 10112010 I Description.. 1 II Gel Selection Guide. 1 III Protein Migration Table.. 2 IV Compatible Gel Boxes... 2 V Related Products. 3 VI Storage.
PROTOCOL Immunostaining for Flow Cytometry 1850 Millrace Drive, Suite 3A Eugene, Oregon 97403 Rev.0 Background The combination of single cell analysis using flow cytometry and the specificity of antibody-based
Protein Precipitation Protocols Notes: All reagents need to high purity/hplc quality. All tubes used should be new or hand cleaned thoroughly with Micro90 detergent. High quality water needs to be used
Lab 5: DNA Fingerprinting You are about to perform a procedure known as DNA fingerprinting. The data obtained may allow you to determine if the samples of DNA that you will be provided with are from the
Name: Biology 309 Lab Notebook This is a guided lab notebook for you to keep well-organized notes about procedures and record experimental data for experiments as they are performed. It is guided because,
Why would you need to purify protein? Methods for Working with Protein 1. Protein Isolation A. Selection of a protein source i. tissue and cell cultures (bacteria, yeast, mammalian, etc.) ii. genetically
In vitro analysis of pri-mirna processing by Drosha-DGCR8 complex (Narry Kim s lab) 1-1. Preparation of radiolabeled pri-mirna transcript The RNA substrate for a cropping reaction can be prepared by in
ReadyPrep Protein Extraction Kit (Cytoplasmic/Nuclear) Instruction Manual Catalog #163-2089 For technical service, call your local Bio-Rad office, or in the US, call 1-800-4BIORAD (1-800-424-6723) Bio-Rad
Odyssey Infrared Imaging System Western Blot Analysis Published January 2003. Revised July, 2007. The most recent version of this protocol is posted at http://biosupport.licor.com/support Doc# 988-09288
Sucrose Gradient Separation Protocol January 2007 CONTENT I. REQUIRED REAGENTS AND EQUIPMENT 3 II. SAMPLE PREPARATION 4 III. SAMPLE SOLUBILIZATION 5 IV. SUCROSE GRADIENT PREPARATION 6 V. CENTRIFUGATION
Introduction to Western Blotting Principles Technical Guidance Data Analysis Troubleshooting Contents Chapter 1 Introduction Overview... 5 Why Use a Western Blot?.... 6 Antibody Considerations... 7 Monoclonals
Rat creatine kinase MM isoenzyme (CK-MM) ELISA Kit Catalog Number. CSB-E14405r For the quantitative determination of rat creatine kinase MM isoenzyme (CK-MM) concentrations in serum, plasma, tissue homogenates.
BIOCHEMICAL FRACTIONATION OF CHLOROPLASTS During the past thirty years, a series of protocols has been developed that permits the homogenization of tissues and the subsequent fractionation and purification
Genomic DNA Extraction Kit INSTRUCTION MANUAL Table of Contents Introduction 3 Kit Components 3 Storage Conditions 4 Recommended Equipment and Reagents 4 Introduction to the Protocol 4 General Overview
Anti-V5 Antibody Anti-V5-HRP Antibody Catalog nos. R960-25, R961-25 Version F 073001 28-0140 www.invitrogen.com email@example.com ii Table of Contents Table of Contents...iii Overview...1 Western
Researcher, 2009;1(2):67-86 Yang and Ma, Western Blotting Method Western Blotting and ELISA Techniques Yan Yang *, Hongbao Ma *, ** * Brookdale University Hospital and Medical Center, Brooklyn, New York
Preparation of Parasite Protein Extracts and Western Blot Analysis Arlett Heiber and Tobias Spielmann * Parasitology Section, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany *For correspondence:
DNA SPOOLING 1 ISOLATION OF DNA FROM ONION INTRODUCTION This laboratory protocol will demonstrate several basic steps required for isolation of chromosomal DNA from cells. To extract the chromosomal DNA,
Bicinchoninic Acid Protein Assay Kit Catalog Numbers BCA1 AND B9643 TECHNICAL BULLETIN Synonym: BCA Product Description Protein determination is one of the most common operations performed in biochemical
EXPERIMENT VI PURIFICATION AND CHARACTERIZATION OF PROTEINS I- Protein isolation and dialysis In order to investigate its structure and properties a protein must be obtained in pure form. Since proteins
SDS-PAGE This lab will introduce you to SDS-PAGE, a simple and inexpensive method for resolving proteins in complex mixtures. SDS-PAGE gels provide the starting materials for western blots and for some
Detection of proteins by lithium dodecyl sulphate polyacrylamide gel electrophoresis During electrophoretic measurements a mixture of compounds in solution is taken into a chamber, two electrodes are joined
Cat. # T7111A For Research Use Western BLoT Product Manual Table of Contents I. Description... 3 II. Components... 3 III. Storage... 3 IV. Materials Required but Not Provided... 3 V. Precautions... 3 VI.
Quick Start Bradford Protein Assay Instruction Manual For technical service call your local Bio-Rad office, or in the US, 1-800-4BIORAD (1-800-424-6723) Table of Contents Section 1 Introduction 1 1.1 Principle
6 Characterization of Casein and Bovine Serum Albumin (BSA) Objectives: A) To separate a mixture of casein and bovine serum albumin B) to characterize these proteins based on their solubilities as a function
Page 1 of 7 Agencourt RNAdvance Cell v2 Total RNA Isolation from Cultured Cells Please refer to http://www.agencourt.com/technical for updated protocols and refer to MSDS instructions http://www.beckmancoulter.com/customersupport/msds/msds.asp