Question

I want you to research how to purify a protein from cell culture. After purification of the protein, you would need to quantify the protein using Bradford Assay and run the protein on SDS-PAGE gel for Western blot analysis to determine the amount of specific in a sample.

1. Write out a protocol for the isolation of protein from human cell culture- you can determine the type of cells you are purifying the protein from
2. Write out a protocol for the Bradford assay including the standard curve and explain how you would determine the amount of protein in your sample from the standard curve.
3. Write out the protocol for the Western Blot analysis making certain to fulling explain:
   1. How an SDS-PAGE gel separates proteins
   2. How proteins are transferred from a SDS PAGE gel to the nitrocellulose filter
   3. Different solutions and their uses
   4. Different antibodies and their uses
   5. How you can visualize the signal and thus your protein

Answer 1

1. Isolation of protein from human cell culture

Procedure

Seed cells into 6 cm diameter plastic dishes. Prior to harvesting, thaw Denaturing Protein Sample Buffer at room temperature and ensure that the SDS becomes fully dissolved. At time of harvesting remove one plate at a time from the incubator and place on ice. Holding the plate in an angle, remove as much medium as possible, using an aspirator. Level the plate, add 4 ml ice-cold PBS and swirl plate quickly. Holding the plate in an angle, remove as much liquid as possible, using an aspirator. Level the plate and add 80 μl Denaturing Protein Sample Buffer to the middle of the plate. Remove cells from the plastic surface immediately using a cell scraper (∼3 cm long blade for 6 cm plastic dish), . For this, move the entire scraper in a circular motion while keeping the angle of the blade the same. At the same time, slowly rotate the plate clockwise, by 360° and repeat the process but rotate plate counter-clockwise. During this procedure the liquid becomes viscous. Holding the plate in an angle, use the scraper to collect the entire sample in the lowest part of the plate. Using a 200 μl pipette, slowly pipette and transfer sample to the bottom of a 1.5 ml microfuge tube. If sample is not in the bottom of the tube, quickly spin in a centrifuge for a few seconds, 4 °C, ∼1000 g. Immediately place sample on dry ice or in liquid nitrogen. If not available, transfer tube to a −80 °C or −20 °C cold metal block, to quickly freeze the sample. Equilibrate shaking heating block to 99 °C and immediatelt transfer frozen samples directly to shaking heating block. Incubate in mixing heating block for 10 min, at 99 °C, with maximum rpm. The samples are ready for denaturing gel electrophoresis. The amount of sample to be loaded on the gel depends on the abundance of the protein of interest, and may require optimization for each specific protein. If not used immediately, samples can be stored at −20 °C or −80 °C until analysis. Prior to loadingtThaw and mix samples very carefully.

2. Bradford assay including the standard curve

Standard bovine serum albumin (BSA)

Typically BSA is used as the standard, since it is cheap and easy to come by. Prepare several dilutions of the BSA standard, at least 5. For example, the dilutions may be 5, 10, 25, 50, 75, and 100 micrograms of BSA per milliliter. Add Bradford reagent (which contains an acid and the Coomassie dye) to the BSA dilutions and incubate for 5 min to 1 hour. Measure absorbance at 595nm with spectrophotometer. The data obtained here can be used to create the standard graph, with the absorbance on the y-axis and the known protein concentration on the x-axis. A fairly linear line should be able to be drawn between each of the points and the equation of the line determined.

Sample

The only difference in Standard and sample analysis is the sample concentration is unknown. Dilute the sample so that it falls within the BSA standard curve and add Bradford reagent. Incubate for 5 min to 1 hour and measure absorbance at 595nm with spectrophotometer. Plug the absorbance of the sample into the equation determined for the standard. Plug the absorbance on the y-axis and the known sample protein concentration on the x-axis.

Volume of Protein Standard (ml) x Starting Protein Concentration= Amount of protein (mg)

3. Protocol for the Western Blot analysis

a) SDS-PAGE separates proteins primarily by mass because the ionic detergent SDS denatures and binds to proteins to make them uniformly negatively charged. Thus, when a current is applied, all SDS-bound proteins in a sample will migrate through the gel toward the positively charged electrode.

b) Proteins must be transferred from the gel onto a suitable membrane for antibody staining and detection after electrophoresis. Transfer is performed by passing a current across the gel to the nitrocellulose filter.

c) Reagents and buffers

• 1x RIPA Buffer: 50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100 or NP40.
• 1x PBS Buffer: 137 mM NaCl, 2.7 mM KCl, 2.7 mM Na2HPO4, 2.7 mM KH2PO4, pH 7.4
• BCA protein assay kit or Bradford protein assay kit
• 1.5 M Tris buffer (pH 8.8): 90.68 g Tris-HCl to ddH2O, adjust pH to 8.8 using HCl and to final 500ml
• 1.0 M Tris buffer (pH 6.8): 60.58 g Tris-HCl to ddH2O, adjust pH to 6.8 using HCl and to final 500ml
• 10% APS: 100 mg AP in 1 ml ddH2O. prepared before use.
• 10% SDS: 10 g SDS in 100 ml ddH2O.
• 1x Tris-Glycine running buffer: 25 mM Tris, 230 mM Glycine (pH 8.3), 0.1% SDS.
• 3x SDS protein loading buffer: 150 mM Tris (pH 6.8), 6% SDS, 30% glycerol, 30 mM EDTA and 0.2% Bromophenol Blue. The buffer should be either freshly prepared or prepared, aliquoted, and frozen for future use.
• 1x TTBS: 25 mM Tris(pH 7.5): 0.15 M NaCl, 0.05% Tween-20, 0.001% Thimerosal
• 1x Transfer Buffer: 3 g Tris, 14.4 g Glycine and 200 ml methanol, add ddH2O to 1L

d) Polyclonal, monoclonal and recombinant secondary antibodies, as well as antibody fragments can be used for western blotting. Polyclonal secondary antibodies are the most common form of secondary antibodies in use.

e) t is visualised through various methods such as staining, immunofluorescence,and radioactivityfor the detection of the specific target protein