Lidstrom:BCA assay: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
Line 16: Line 16:


==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 Kit.jpg|thumb|upright=3.0|center|Preparing BSA standards for BCA total protein assay]]
## [[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:
Line 23: Line 26:
##[[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 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
#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

  1. Plan & Prepare your samples & dilutions
    1. 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.
    2. You will need 25 uL of each sample per well. Consider doing technical replicates.
  2. 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
  3. 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)
  4. Prepare your Microplate
    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

References

Contact

or instead, discuss this protocol.

Retrieved from "https://openwetware.org/mediawiki/index.php?title=Lidstrom:BCA_assay&oldid=699903"