Lidstrom:BCA assay
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Overview
- BCA is a general use kit to determine total protein concentration prior to Western blot or other protein analysis
- Our kit: Pierce BCA Protein Assay Kit (Prod #23225). MANUAL
- Use the microplate procedure
- Use the buffer your samples are dissolved in for the diluent.
Materials
- BCA reagent A
- BCA reagent B
- 96 well plate
- Microcentrifuge tubes
- Microcentrifuge tube rack
- Microcentrifuge
- BSA stock (2 µg/ µl)
- Pipette
- Pipette tips
Procedure
- ?? Do you need to turn on the plate reader and warm up the lamp??
- Plan & Prepare your samples & dilutions
- Make three concentrations of each of in sets of three new microcentrifuge tubes labeled with their dilution or tube number. Full concentration, 2x dilution and 10x dilutions are usually sufficient.
- You will need 25 uL of each sample per well. Consider doing technical replicates.
- Prepare Standards ( 2 fold dilutions): 2 µg/ µl, 1 µg/ µl, 0.5 µg/ µl, 0.25 µg/ µl, 0.125 µg/ µl
- You need at least 1mL of the full-concentration sample (A) to do the dilutions below, so weigh at least 2ug in an eppendorf tube.
- Prepare BCA Working Reagent
- For the total volume of working reagent calculate:
- (# standards (in our case 5) and samples (30 in our case))*(# replicates (2))*(volume of working solution per sample (200 µl))
- To prepare working solution mix 50 parts Reagent A with 1 part Reagent B (ie. 50 ml Reagent A plus 1 ml Reagent B)
- For the total volume of working reagent calculate:
- Prepare your Microplate
- Pipette 25 µl of each standard or unknown sample replicate into the designated microplate well
- Add 200 µl of the WR to each well and mix plate thoroughly on a plate shaker for 30 seconds
- Incubate plate at 37C for 30 minutes
- Remove plate and measure the absorbance at 562 nm on a plate reader
- Subtract the average 562 nm absorbance measurement of the Blank standard replicates from the 562 absorbance of al the individual standard and unknown
- Prepare a standard curve by plotting the average Blank=corrected 562 nm measurement for each BSA standard vs. its concentration in µg/µl. Use the standard curve to determine the protein concentration of each unknown sample
Re-using standards
- One way to save time would be to re-use standard samples you had prepared previously. Amanda smith has tested samples that were stored in the fridge (hence no freeze-thaw cycles) for several weeks and they worked the same.
Contact
Page set up by Janet 5/2013