Lidstrom:BCA assay: Difference between revisions
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==Procedure== | ==Procedure== | ||
#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 | # Prepare Standards ( 2 fold dilutions): 2 µg/ µl, 1 µg/ µl, 0.5 µg/ µl, 0.25 µg/ µl, 0.125 µg/ µl | ||
## [[image:Prepare BCA Standards for Pierce | ## [[image:Prepare BCA Standards for Pierce kit.jpg|thumb|upright=3.0|center|Preparing BSA standards for BCA total protein assay]] | ||
#Prepare BCA Working Reagent | #Prepare BCA Working Reagent | ||
##For the total volume of working reagent calculate: | ##For the total volume of working reagent calculate: | ||
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##[[image:Prepare working reagent (WR) standards for Pierce kit.jpg|thumb|upright=3.0|center|Prepare working reagent (WR) standards for Pierce kit]] | ##[[image:Prepare working reagent (WR) standards for Pierce kit.jpg|thumb|upright=3.0|center|Prepare working reagent (WR) standards for Pierce kit]] | ||
##To prepare working solution mix 50 parts Reagent A with 1 part Reagent B (ie. 50 ml Reagent A plus 1 ml Reagent B) | ##To prepare working solution mix 50 parts Reagent A with 1 part Reagent B (ie. 50 ml Reagent A plus 1 ml Reagent B) | ||
#Prepare your Microplate | #Prepare your Microplate | ||
##Pipette 25 µl of each standard or unknown sample replicate into the designated microplate well | ##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 | ##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 | ##Incubate plate at 37C for 30 minutes |
Revision as of 13:31, 27 May 2013
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
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
- 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
- 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
Notes
Used at Stanford for Tissue Engineering Lab Course (ME385B and 2007 Winter/Summer TC Workshops
References
Contact
or instead, discuss this protocol.