Protein Detection Methods & Western Blots

Transcription

1 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 one protein out of complex mixture

2 What is SDS-PAGE? A procedure to separate proteins and determine their Molecular Weights.

3 Theory Behind Electrophoresis Charged molecules in an electric field behave in a predictable manner. Positively charged molecules will move towards the negative pole while negatively charged molecules move towards the positive pole. Movement of any charged species through an electric field is determined by it s net charge, molecular radius and the magnitude of the applied field, but not size.

4 How do we get proteins to separate by size? Need to disrupt tertiary structure Make charge uniform

5 The importance of SDS SDS is a negatively charged (anionic) detergent. Coats proteins and disrupts secondary and tertiary protein structures by breaking hydrogen bonds and unfolding or denaturing protein. Masks charge on protein by imparting a large negative charge so that all proteins act the same in regards to charge. Prevents protein aggregation. Prevents protein shape from influencing gel run. Size of protein determines migration in gel.

6 SDS linearizesproteins bitesizebio.com All proteins have the same mass to charge ratio Have the same molecular radius Thus, migration through a matrix is determined by length which is proportional to molecular weight

7 SDS-PAGE: The Matrix Free radicals bitesizebio.com Transfer of electrons to acrylamideand bisacrylamide; causing them to react Amount of crosslinking, thus pore size and consequent separation properties of the gel can be controlled by varying the ratio of acrylamide to bis-acrylamide

8 SDS-PAGE: The buffer system Discontinuous Laemmlibuffer system -buffer in the gel and the tank are different Gly proteins Cl - Stacking gel: 4% Resolving gel: range 5-20% bitesizebio.com Proteins are concentrated into the narrow zone between the Cl - and glycinefronts.

9 Polyacrylamideconcentration alters resolution of protein migration

10 Uses of SDS-PAGE Determine protein size Identify protein Determine sample purity Identify existence of disulfide bonds Quantify amounts of protein

11 Detection of protein after SDS-PAGE STAINING Technique-The goal of staining is to bind a chromaphoreto a polypeptide Not Quantitative-All stains are very qualitative, individual polypeptides differ greatly in their ability to bind a stain Low Reproducibility-Different staining techniques will not stain a polypeptide consistently Range-There is a small concentration range over which a particular stains is useful Non-Specific Binds to majority of proteins; unable to determine if your protein of interest is in the protein extract run on the gel

12 Protein dyes used for staining Stain Advantages Disadvantages Coomassie Blue Simple methodology MS compatible Easy to image Very insensitive Bio-Safe Colloidal Coomassie Stain Silver stain Sliver stain Plus Ruby stain Simple methodology MS compatible More sensitive Easy to image Very sensitive Easy to image Very sensitive Easy to image More MS compatible than standard silver staining Very sensitive MS compatible Quantitative over 3 orders of magnitude Simple methodology Less sensitive than silver Complex procedure Incompatible with MS Not quantitative Complex procedure Not very MS compatible Not quantitative Difficult to image

13 Other Methods of Protein Detection 1.Labeling proteins with radioisotopes during expression (e.g. 35 S-methionine) 1.Western blotting (e.g. antibody against protein of interest)

14 Bio-Rad s take on Western blotting! Comparative Proteomics Kit II: Western Blot Module

15 According to Bio-Rad Powerful teaching tool Why Teach Western Blotting? Real-world connections (?) Laboratory extensions (?) Tangible results Link to careers and industry (?) Standards-based (?)

16 Why use Western blotting? Can specifically determine if your protein of interest is present in a crude protein preparation

17 Transfer Proteins from the gel to the nitrocellulose membrane Protein Transfer 60 minutes 200 ma Blotting buffer 1x Tris glycine with 20% methanol, 0.1% SDS Electric Current

18 Blocking membrane before addition of antibody Blocking Buffer Remove membrane from the blotting sandwich and immerse in blocking solution 5% non-fat milk or 1% gelatin: Prevents the primary antibody from binding randomly to the membrane Trisor Phosphate buffered saline (TBS or PBS): Provides the correct environment (ph, Salt) to maintain protein shape 0.025% Tween20: non-ionic detergent that prevents non-specific binding of antibodies to the membrane; TBS-T or PBS-T when added to buffer

19 Antibodies used for Western Molecule of interest is injected into primary animal model Animal makes antibodies against the molecule Antibodies are purified (primary antibody)

20 Addition of the 1 o antibody Discard blocking solution Pour primary antibody (in TBST) onto the membrane and gently rock for 30 minutes Primary antibody will bind to the histidinetag Add 50ml of wash (TBST) rock for 10 min to wash

21 Addition of 2 o Antibody Discard wash solution Pour secondary antibody onto the membrane and gently rock for 30 minutes Secondary antibody will bind to the primary antibody Quickly rinse membrane in 50ml of wash buffer and discard the wash buffer Add 50ml of wash leave for 3 minutes on the rocking platform

22 Production of 2 o Antibody Antibodies from the first animal model are injected into a second animal model The second animal produces antibodies against the first antibody (secondary antibody) The secondary antibody is purified and conjugated to a colorimetric substrate or to an enzyme that can cleave a colorimetric compound

23 Detection of Protein Discard wash solution Add the enzyme substrate (HRP color detection reagent) to the membrane Incubate for 10 minutes The colorimetric substrate is cleaved by the enzyme conjugated (attached) to the secondary antibody

24 Other detection methods Chemiluminescent substrates Film or cooled CCD camera Infrared emitting side groups Special machine Quantum dots EpiUV and CCD camera with EtBRfilter

25 Western vselisa Enzyme-Linked Immunosorbant Assay ELISA - Quick results - Primary screening - Identifies proteins by antibody specificity only -With correct controls can be quantitative Western Blot - Confirm ELISA results - More specific -Identifies proteins by both antibody specificity and size -Difficult to use for quantification

26 Questions?