Lidstrom:BCA assay: Difference between revisions
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(New page: ==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). [http:...) |
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##For the total volume of working reagent calculate: | ##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)) | ##*(# standards (in our case 5) and samples (30 in our case))*(# replicates (2))*(volume of working solution per sample (200 µl)) | ||
##[[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 samples | #Prepare your samples | ||
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##Remove plate and measure the absorbance at 562 nm on a plate reader | ##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 | ##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 | ##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== | ==Notes== |
Revision as of 13:25, 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
- 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 samples
- Make three concentrations of your samples in three new microcentrifuge tubes labeled S1, S2, S3(to ensure you get within the range of 0.125-2 µl)
- Put 60 µl of your sample in tube S1
- Make a 1:2 dilution (30 µl sample + 30 µl RIPA buffer) in S2
- Make a 1:10 dilution ( 10 µl sample + 90 µl RIPA buffer) in S3
- Make three concentrations of your samples in three new microcentrifuge tubes labeled S1, S2, S3(to ensure you get within the range of 0.125-2 µl)
- 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.