- Table of Contents
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Choosing the correct protein extraction method is crucial, ultimately making the difference between a blank blot and a beautiful blot. To extract protein for the western blot analysis, the first step is to prepare for the lysate from samples. Cell culture and tissue are the most common samples to start with.Learn how to optimize WB sample preparation
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|Tube||Diluent Volume (µL)||BSA Volume (µL)||BSA Concentration (µg/mL)|
|A||0||600 (From Tube A)||2000|
|B||100||300 (From Tube A)||1500|
|C||300||300 (From Tube A)||1000|
|D||200||200 (From Tube B)||750|
|E||300||300 (From Tube C)||500|
|F||300||300 (From Tube E)||250|
|G||300||300 (From Tube F)||125|
|H||400||100 (From Tube G)||25|
Note: Increasing the incubation time and temperature can increase the net 562 nm absorbance for each test and decrease both the minimum detection level of the reagent and the working range of the protocol. Use a water bath to heat the tubes for either Standard (37°C incubation) or Enhanced (60°C incubation) Protocol. Using a forced-air incubator can introduce significant error in color development because of uneven heat transfer.
Note: Because the BCA Assay does not reach a true end point, color development will continue even after cooling to room temperature. However, because the rate of color development is low at room temperature, no significant error will be introduced if the 562 nm absorbance measurements of all tubes are made within 10 min of each other.
Subtract the average 562 nm absorbance measurement of the blank standard replicates from the 562 nm absorbance measurement of all other individual standard and unknown sample replicates.
Prepare a standard curve by plotting the average blank-corrected 562 nm measurement for each BSA standard vs. its concentration in μg/mL. Use the standard curve to determine the protein concentration of each unknown sample.
Learn the concept behind Western blotting. It is a technique that is used to detect specific proteins in the given sample. It usually involves two major processes, namely, SDS-polyacrylamide gel electrophoresis and protein blotting and testing.Learn Western Blot Principle
Get to learn the concept behind our best practises on Western Blot optimization. Learn how to optimize every aspect of your experiment to yield the best results.Learn Western Blot optimization
Get to learn Pro tips on resolving common Western Blot issues such as weak signal, wrong band size, smiley gel, and high background.Browse Western Blot Troubleshooting Tips