Protein assay. Absorbance Fluorescence Emission Colorimetric detection BIO/MDT 325. Absorbance

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1 Protein assay Absorbance Fluorescence Emission Colorimetric detection BIO/MDT 325 Absorbance Using A280 to Determine Protein Concentration Determination of protein concentration by measuring absorbance at 280 nm (A280) is based on the absorbance of UV light by aromatic amino acids in protein solutions due primarily to tryptophan and tyrosine residues and to a lesser extent phenylalanine residues. The measured absorbance of a protein sample solution is used to calculate the concentration either from its published absorptivity at 280 nm (A280) or by comparison with a calibration curve prepared from measurements with standard protein solutions. This assay can be used to quantitate solutions with protein concentrations of 20 to 3000 µg/ml. 1

2 Using A205 to Determine Protein Concentration Determination of protein concentration by measurement of absorbance at 205 nm (A205) is based on absorbance by the peptide bond. The concentration of a protein sample is determined from the measured absorbance and the absorptivity at 205 nm (A205). This assay can be used to quantitate protein solutions with concentrations of 1 to 100 µg/ml protein. Fluorescence Emission Using Fluorescence Emission to Determine Protein Concentration Protein concentration can also be determined by measuring the intrinsic fluorescence based on fluorescence emission by the aromatic amino acids tryptophan, tyrosine, and/or phenylalanine. Usually tryptophan fluorescence is measured. The fluorescence intensity of the protein sample solution is measured, and the concentration of the protein sample solution calculated from a calibration curve based on the fluorescence emission of standard solutions prepared from the purified protein. This assay can be used to quantitate protein solutions with concentrations of 5 to 50 µg/ml. Measurement of intrinsic fluorescence by aromatic amino acids is primarily used to obtain qualitative information 2

3 Colorimetric detection Protein assay reagents involve either protein-dye binding (coomassie) chemistry, protein-copper chelation chemistry, and protein-phenol chemistry. protein-dye binding Using the Bradford Method to Determine Protein Concentration The Bradford method depends on quantitating the binding of a dye, Coomassie brilliant blue, to an unknown protein and comparing this binding to that of different amounts of a standard protein, usually BSA. It is designed to quantify 1 to 10 µg protein. Protein determinations in the range of 10 to 100 µg may be carried out by increasing the volume of the dye solution 5-fold and using larger tubes. 3

4 protein-phenol chemistry Using the Lowry Method to Determine Protein Concentration The Lowry method depends on quantitating the color obtained from the reaction of Folin-Ciocalteu phenol reagent with the tyrosyl residues of an unknown protein and comparing this color value to the color values derived from a standard curve of a standard protein, usually BSA. This assay is designed to quantify 1 to 20 µg protein. Protein determinations in the range of 5 to 100 µg may be carried out by increasing all the volumes 5-fold. protein-copper chelation chemistry Pierce Micro BCA protein assay Colorimetric method; read at 562 nm Compatible with most ionic and nonionic detergents A very sensitive reagent for dilute protein samples Linear working range for BSA: µg/ml 4

5 BCA assay Use BCA (bicinchoninic acid) as the detection reagent for cuprous ion (Cu +1 ), which is formed when Cu +2 is reduced by protein in an alkaline environment. A purple-colored reaction product is form by chelation of two molecules of BCA with one cuprous ion. This complex exhibits a strong absorbance at 562 nm. The number of peptide bonds and the presence of 3 amino acids (cysteine, tryptophan and tyrosine) are responsible for color formation with BCA. 1. Prepare diluted BSA standards 2. Prepare Micro BCA working reagent (WR) 3. Follow test tube procedure 4. Determine the concentration of Protein A, B, and C. 5

6 A. Preparation of Diluted Albumin (BSA) Standards Vial Volume of Diluent Volume and Source of BSA Final BSA Concentration A 4500 µl 500 µl of Stock 200 µg/ml B 8000 µl 2000 µl of vial A dilution 40 µg/ml C 4000 µl 4000 µl of vial B dilution 20 µg/ml D 4000 µl 4000 µl of vial C dilution 10 µg/ml E 4000 µl 4000 µl of vial D dilution 5 µg/ml F 4000 µl 4000 µl of vial E dilution 2.5 µg/ml G 4800 µl 3200 µl of vial F dilution 1 µg/ml H 4000 µl 4000 µl of vial G dilution 0.5 µg/ml I 8000 µl 0 0 µg/ml = Blank 6

7 B. Preparation of the Working Reagent (WR) 1. For the standard Test Tube Procedure with 3 unknowns : (9 standards + 3 unknowns) (2 ml) = 24 ml WR required (round up to 25 ml) 2. Prepare fresh WR by mixing 25 parts of Micro BCA Reagent MA and 24 parts Reagent MB with 1 part of Reagent MC (25:24:1, Reagent MA:MB:MC). For our experiment, combine 12.5 ml of Reagent MA and 12.0 ml Reagent MB with 0.5 ml of Reagent MC. 1. Pipette 2.0 ml of each standard and unknown sample into an appropriately labeled test tube. 2. Add 2.0 ml of the WR to each tube and mix well. 3. Cover tubes and incubate at 60 C in a water bath for 1 hour. 4. Cool all tubes to RT. 5. With the spectrophotometer set to 562 nm, zero the instrument on a cuvette filled only with water. Subsequently, measure the absorbance of all the samples within 10 minutes 6. Subtract the average 562 nm absorbance reading of the Blank standard replicates from the 562 nm reading of all other individual standard and unknown sample replicates. 7. Prepare a standard curve by plotting the average Blank-corrected 562 nm reading for each BSA standard vs. its concentration in µg/ml. Use the standard curve to determine the protein concentration of each unknown sample. 7

8 2. Modified Lowry Protein Assay The most widely cited colorimetric method; read at 750 nm Linear results from 1-1,500 µg/ml for BSA Convenient microplate or cuvette format Less protein-to-protein variation than dye-binding methods 1. Prepare diluted BSA standards 2. Prepare reagent 3. Follow test tube procedure 4. Determine the concentration of Protein A, B, and C. 8

9 A. Preparation of Diluted Albumin (BSA) Standards Vial Volume of Diluent Volume and Source of BSA Final BSA Concentration A 250 µl 750 µl of Stock 1,500 µg/ml B 625 µl 625 µl of Stock 1,000 µg/ml C 310 µl 310 µl of vial A dilution 750 µg/ml D 625 µl 625 µl of vial B dilution 500 µg/ml E 625 µl 625 µl of vial D dilution 250 µg/ml F 625 µl 625 µl of vial E dilution 125 µg/ml G 800 µl 200 µl of vial F dilution 25 µg/ml H 800 µl 200 µl of vial G dilution 5 µg/ml I 800 µl 200 µl of vial H dilution 1 µg/ml J 1000 µl 0 0 µg/ml = Blank 9

10 B. Preparation of 1X Folin-Ciocalteu Reagent Prepare 1X (1 N) Folin-Ciocalteu Reagent by diluting the supplied 2X (2 N) reagent 1:1 with ultrapure water. [2.1 ml of 2x reagent and 2.1 ml of water] Each test replicate requires 300 µl of 1X Folin-Ciocalteu Reagent in the Test Tube Protocol. 1. Pipette 0.6 ml of each standard and unknown sample replicate into an appropriately labeled test tube. 2. At 15-second intervals, add 3.0 ml of Modified Lowry Reagent to each test tube. Mix well and incubate each tube at room temperature (RT) for exactly 10 minutes. 3. Exactly at the end of each tube s 10-minute incubation period, add 300 µl of prepared 1X Folin-Ciocalteu Reagent, immediately vortex to mix the contents. Maintain the 15-second interval between tubes established in Step Cover and incubate all tubes at RT for 30 minutes. 5. With the spectrophotometer set to 750 nm, zero the instrument on a cuvette filled only with water. Subsequently, measure the absorbance of all the samples. 6. Subtract the average 750 nm absorbance values of the Blank standard replicates from the 750 nm absorbance values of all other individual standard and unknown sample replicates. 7. Prepare a standard curve by plotting the average Blank-corrected 750 nm value for each BSA standard vs. its concentration in µg/ml. Use the standard curve to determine the protein concentration of each unknown sample. 10

11 pglo The cloned DNA fragment carries a gene that encodes a Green Fluorescent Protein (GFP). When GFP is illuminated with long wave ultraviolet (UV) light it gives off a bright green fluorescence, and as a result, cells that produce large amounts of GFP glow bright green when irradiated by long wave UV. Plasmid: a circular DNA molecule (cloning vector), capable of self-replicating, carrying one or more genes for antibiotic resistance protein and a cloned foreign gene. pglo: GFP gene is under control of the arabinose operon A restriction endonuclease map of pglo. The arrows on the circle represent genes or promoter sequences and the direction of their transcription. Abbreviations: ori, origin of replication; bla, betalactamase gene; gfp, green fluorescent protein gene; P BAD, promoter sequence of the E. coli arabad operon. 11

12 Regulation of ara operon On pglo, the promoter sequence controlling gfp expression usually regulates the transcription of a group of genes in E. coli called the ara operon. These genes encode proteins needed to metabolize the sugar L-arabinose. Before RNA polymerase can bind effectively to P BAD two events must occur, first the arabinose-arac complex must bind to arai just upstream of the promoter and second the camp-cap complex must bind to a sequence just upstream from arai. As long as arabinose is the only carbon source available to E. coli, transcription from P BAD occurs. No (or very little) transcription occurs from the ara operon promoter (also called P BAD ) unless the activator protein AraC first binds to a specific activator sequence (arai) just upstream of P BAD. AraC is thought to help RNA polymerase bind to P BAD and initiate transcription. However, AraC only binds to the appropriate activator sequence in the presence of the sugar arabinose. AraC is an allosteric protein with two binding sites, the first binds arabinose causing a conformational change in the protein that allows the second site to recognize and bind to arai. Thus, in the absence of arabinose, P BAD is effectively switched off, whereas in its presence the promoter is active. This process is called positive regulation because it requires the involvement of an activator protein that only binds to a specific activator sequence in the presence of an effector molecule, in this case arabinose, making transcription possible. In addition to the positive control exerted by AraC, efficient transcription from P BAD is dependent on another form of positive regulation mediated by the camp-cap complex. 12

13 In the presence of glucose If another more preferred carbon source, such as glucose, is available transcription from P BAD is significantly reduced. High levels of glucose inhibit the synthesis of cyclic adenosine monophosphate or cyclic AMP (camp), and without camp, the catabolite activator protein (CAP) cannot bind to its target sequence. Without the camp-cap complex, RNA polymerase cannot bind efficiently to P BAD even in the presence of the arabinose-arac complex and thus, very little transcription occurs. This effect is referred to as catabolite repression, a global regulatory mechanism that controls the expression of many operons in E. coli, including the well-characterized lac operon. 13