# Lidstrom:BCA assay

(Difference between revisions)
 Revision as of 16:25, 27 May 2013 (view source) (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:...)← Previous diff Revision as of 16:25, 27 May 2013 (view source) (→Procedure)Next diff → Line 21: Line 21: ##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 Line 33: Line 34: ##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==

## 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

1. Prepare Standards ( 2 fold dilutions): 2 µg/ µl, 1 µg/ µl, 0.5 µg/ µl, 0.25 µg/ µl, 0.125 µg/ µl
1. Preparing BSA standards for BCA total protein assay
2. Prepare BCA Working Reagent
1. 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))
2. Prepare working reagent (WR) standards for Pierce kit
3. To prepare working solution mix 50 parts Reagent A with 1 part Reagent B (ie. 50 ml Reagent A plus 1 ml Reagent B)
1. 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)
1. Put 60 µl of your sample in tube S1
2. Make a 1:2 dilution (30 µl sample + 30 µl RIPA buffer) in S2
3. Make a 1:10 dilution ( 10 µl sample + 90 µl RIPA buffer) in S3
1. Pipette 25 µl of each standard or unknown sample replicate into the designated microplate well
2. Add 200 µl of the WR to each well and mix plate thoroughly on a plate shaker for 30 seconds
3. Incubate plate at 37C for 30 minutes
4. Remove plate and measure the absorbance at 562 nm on a plate reader
5. Subtract the average 562 nm absorbance measurement of the Blank standard replicates from the 562 absorbance of al the individual standard and unknown
6. 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