Jacobs:Protocol BCA Total Protein

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'''Click the view source tab and copy everything below this line.  Paste it into your new protocol page.  Then replace the text in this page with your own protocol.  Feel free to add or delete sections as appropriate.'''
 
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==Overview==
==Overview==
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Replace this sentence with a brief description of the protocol and its goal.
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BCA is a general use kit to determine total protein concentration prior to Western blot or other protein analysis
==Materials==
==Materials==
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List reagents, supplies and equipment necessary to perform the protocol here.  For those materials which have their own OWW pages, link to that page.  Alternatively, links to the suppliers' page on that material are also appropriate.
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* BCA reagent A
 +
* BCA reagent B
 +
* 96 well plate
 +
* Microcentrifuge tubes
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* Microcentrifuge tube rack
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* Microcentrifuge
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* BSA stock (2 µg/ µl)
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* Pipette
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* Pipette tips
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*supply 1 (i.e. tubes of a certain size? spreaders?)
 
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*reagent 1
 
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*X &mu;L reagent 2
 
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**component A (reagent 2 is made up of multiple components)
 
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**component B
 
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*equipment 1
 
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*equipment 2
 
==Procedure==
==Procedure==
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#Step 1
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# Prepare Standards ( 2 fold dilutions): 2 µg/ µl, 1 µg/ µl, 0.5 µg/ µl, 0.25 µg/ µl, 0.125 µg/ µl
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#Step 2
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## Label 5 microcentrifuge tubes 1-5 (1= highest concentration, 5= lowest concentration)
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#*Step 2 has some additional information that goes with it.  i.e. Keep at 4&deg;C.
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##In tube #1, put in 120 µl of BSA, in all other tubes put in 60 µl of RIPA lysis Buffer (you always want to use the same dilutant material as what you used to isolate your protein)
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#Step 3
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##Pipette 60 µl of BSA from tube #1 into #2.  Pipette up and down a dozen times or until you think it is properly mixedThen take 60 µl from tube #2 and put it in tube #3 and pipette up and down. Continue this process through tube #5 (this will leave tube #5 with 120 µl)
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##Step 3 has multiple sub-steps within it.
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#Prepare BCA Working Reagent
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##Enumerate each of those.
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##For the total volume of working reagent calculate:
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##*(# 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)
 +
#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
 +
##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==
==Notes==
 +
Used at Stanford for Tissue Engineering Lab Course (ME385B and 2007 Winter/Summer TC Workshops
 +
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Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
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==References==
==References==
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'''Relevant papers and books'''
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<!--'''Relevant papers and books'''
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<!-- If this protocol has papers or books associated with it, list those references here.  See the [[OpenWetWare:Biblio]] page for more information. -->
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If this protocol has papers or books associated with it, list those references here.  See the [[OpenWetWare:Biblio]] page for more information.  
<biblio>
<biblio>
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#Goldbeter-PNAS-1981 pmid=6947258
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#Jacob-JMB-1961 pmid=13718526
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</biblio>-->
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#Ptashne-Genetic-Switch isbn=0879697164
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</biblio>
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==Contact==
==Contact==
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*Who has experience with this protocol?
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*Originally prepared by CRJ-EJC 1/3/06 
 +
 
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].  
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].  
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[[Category:Protocol]]
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[[Category:Needs attention]] <!--Delete this line once the protocol is complete-->
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[[Category:Protein]]
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Revision as of 17:48, 24 April 2008

Contents

Overview

BCA is a general use kit to determine total protein concentration prior to Western blot or other protein analysis

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. Label 5 microcentrifuge tubes 1-5 (1= highest concentration, 5= lowest concentration)
    2. In tube #1, put in 120 µl of BSA, in all other tubes put in 60 µl of RIPA lysis Buffer (you always want to use the same dilutant material as what you used to isolate your protein)
    3. Pipette 60 µl of BSA from tube #1 into #2. Pipette up and down a dozen times or until you think it is properly mixed. Then take 60 µl from tube #2 and put it in tube #3 and pipette up and down. Continue this process through tube #5 (this will leave tube #5 with 120 µl)
  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. To prepare working solution mix 50 parts Reagent A with 1 part Reagent B (ie. 50 ml Reagent A plus 1 ml Reagent B)
  3. Prepare your samples
    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
  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

  • Originally prepared by CRJ-EJC 1/3/06


or instead, discuss this protocol.

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