Jacobs:Protocol BCA Total Protein: Difference between revisions
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==Overview== | ==Overview== | ||
BCA is a general use kit to determine total protein concentration prior to Western blot or other protein analysis | |||
==Materials== | ==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== | ==Procedure== | ||
# | # Prepare Standards ( 2 fold dilutions): 2 µg/ µl, 1 µg/ µl, 0.5 µg/ µl, 0.25 µg/ µl, 0.125 µg/ µl | ||
# | ## Label 5 microcentrifuge tubes 1-5 (1= highest concentration, 5= lowest concentration) | ||
# | ##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) | ||
# | ##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) | ||
## | #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) | |||
#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== | ||
<!-- | |||
#List troubleshooting tips here. | #List troubleshooting tips here. | ||
#You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites. | #You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites. | ||
| Line 43: | Line 53: | ||
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. | ||
--> | |||
==References== | ==References== | ||
'''Relevant papers and books''' | <!--'''Relevant papers and books''' | ||
If this protocol has papers or books associated with it, list those references here. See the [[OpenWetWare:Biblio]] page for more information. | |||
<biblio> | <biblio> | ||
</biblio>--> | |||
</biblio> | |||
==Contact== | ==Contact== | ||
* | *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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<!-- You can tag this protocol with various categories. See the [[Categories]] page for more information. --> | <!-- You can tag this protocol with various categories. See the [[Categories]] page for more information. --> | ||
[[Category:Protocol]] | [[Category:Protocol]] | ||
[[Category: | [[Category:Protein]] | ||
<!-- Move the relevant categories above this line to tag your protocol with the label | <!-- Move the relevant categories above this line to tag your protocol with the label | ||
[[Category:In vitro]] | [[Category:In vitro]] | ||
[[Category:In vivo]] | [[Category:In vivo]] | ||
[[Category:DNA]] | [[Category:DNA]] | ||
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[[Category:Escherichia coli]] | [[Category:Escherichia coli]] | ||
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Revision as of 15:40, 24 April 2008
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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
- Prepare Standards ( 2 fold dilutions): 2 µg/ µl, 1 µg/ µl, 0.5 µg/ µl, 0.25 µg/ µl, 0.125 µg/ µl
- Label 5 microcentrifuge tubes 1-5 (1= highest concentration, 5= lowest concentration)
- 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)
- 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)
- 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
References
Contact
- Originally prepared by CRJ-EJC 1/3/06
or instead, discuss this protocol.
