Getting Started
You have samples and you want to see how much protein is in them. You can use this protocol to quantitate protein in your samples as long as your samples have small amounts of imidazole (≤ 50 mM).
Materials and Equipment
Protocol
Make a BSA protein standard.
Take nine (9) PCR tubes and label them label A-I.
Add 150 uL of sample buffer (i.e., whatever buffer your protein is in, e.g., P1 Buffer) to tubes B-I.
Add 300 uL of Bovine serum albumin (BSA) at 2 mg / mL to tube A.
Using a fresh pipette tip for each transfer, perform a serial dilution. Transfer 150 uL from tube A to tube B and mix by pipetting. Then, transfer 150 uL from tube B to tube C. Continue until you reach tube H. DO NOT add protein to tube I (negative)!
Make BCA working solution.
Calculate how much working solution you need. Prepare at least 200 uL per reaction, with nine (9) standards plus X experimental samples.
Add BCA solution to Cu solution at 50:1 in a 15 mL centrifuge tube (e.g., 2.5 mL BCA + 50 uL Cu solution).
Assemble BCA reactions
For each sample (including the BSA protein standard), add 20 uL of sample to a fresh PCR tube. Label the tube.
Using a multichannel pipette, add 200 uL of working solution to each tube simultaneously. Your reactions are now proceeding slowly.
Incubate BCA reactions @37C / 30 min.
Measure absorbance and analyze data.
Load a 96-well plate with 200 uL of each reaction per well.
Using a plate reader, read absorbance at 560 nm.
Blank samples: subtract the measured absorbance of the blank (tube I) from the measured absorbances for each other well.
Make a calibration curve from absorbance values from the BSA protein standard.
Interpolate your sample concentrations from your calibration curve.
Resources and References
Credits
Yan Zhang, Zoila Jurado, and Miki Yun (Richard Murray Lab, Caltech)
- Developers
