Western Blot is a common method to detect and analyze proteins. It is built on a technique that involves transferring, also known as blotting, proteins separated by electrophoresis from the gel to a membrane where they can be visualized specifically. It is often used in research to separate and identify proteins. In this technique a mixture of proteins is separated based on molecular weight, and thus by type, through gel electrophoresis. These results are then transferred to a membrane producing a band for each protein.
There are nine steps in the whole process of western blot. They are Protein Sample Preparation, Protein Quantification, Protein Sample Preparation, SDS-PAGE, Transfer, Blocking, Primary Antibody Incubation, Secondary Antibody Incubation, Image Development.
You may get a few problems in every step. But people asked most is how to calculate protein concentration for Western Blot. It is in the step of Protein Quantification. You need to make standard curve drawing with Bradford Method. Here are the details.
- Prepare bovine serum albumin (BSA) standard:
Dissolve 0.05 g BSA in 5 mL PBS to obtain 10 mg/mL BSA standard.
- Prepare Coomassie Brilliant Blue G-250 staining solution:
Dissolve 50 mg Coomassie Brilliant Blue G-250 in 25 mL 90% ethanol, add 50 mL phosphoric acid (85%) , and dilute with pure water to 500 mL. Keep away from light.
- Dilute protein samples:
Perform 1:10, 1:20, and 1:40 dilutions for protein samples.
- Dilute BSA standard to the following concentrations.
0; 0.05; 0.075; 0.1; 0.15; 0.2; 0.3; 0.4
Add 20 μL each of diluted BSA standard solution and protein sample to the wells of microplate strip. Then add 180 μL of G250 staining solution to each well and mix thoroughly.
Measure absorbance with spectrophotometer at a wavelength of 595 nm and make a standard curve to calculate the protein concentration.
Well, it is only a part of Cusabio Western Blot Protocol. You can also find the completed version and read it.
In addition, in the protein sample preparation part. You may need a tris buffer calculator, and Cusabio has developed some research tools for you.
A word of warning. A number of tests have been developed along this line. This includes the lowry, bradford, LEAP (latex Elisa for allergenic proteins), and others. All have had interference problems ( false positives is one common one). Modifications to the methods come out periodically, which are more or less successfull. Be aware of possible interferences.
A related field is BSE and prions, if anyone is intetested.
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