Lidstrom:BCA assay

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(Re-using standards)
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Back to [[Lidstrom:Protocols|Protocols]]
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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
*BCA is a general use kit to determine total protein concentration prior to Western blot or other protein analysis
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* BCA reagent B
* BCA reagent B
* 96 well plate
* 96 well plate
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** Use BD Falcon plate (VWR item # [https://us.vwr.com/store/catalog/product.jsp?product_id=4949779 29442-070]) that is 100% clear and has flat bottoms.  The list price is ~$1.75/plate.  You don't need sterile plates or plates that are treated for tissue culture; both are more expensive.  Polystyrine works well, vinyl may be sensitive to more chemicals like phenol/chloroform.     
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** They are kept above the old GC
* Microcentrifuge tubes
* Microcentrifuge tubes
* Microcentrifuge tube rack
* Microcentrifuge tube rack
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* Pipette  
* Pipette  
* Pipette tips
* Pipette tips
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* Plate reader.  Can be either the [[Lidstrom: Tecan Plate Reader|Tecan]] (dark colored) or the [[Lidstrom: Molecular Devices Plate Reader|Molecular devices]] (white)
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== Tips ==
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* Don't wait a whole 30 min before measuring the concentrations in the plate reader.  The standards show a linear relationship within 5-10 min.  Ceci always reads the plate within 5-10 min.
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* Either plate reader can be used.  The Tecan has a 540nm filter for OD measurements.
==Procedure==
==Procedure==
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== Re-using standards==
== Re-using standards==
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* One way to save time would be to re-use standard samples you had prepared previously.  Amanda smith has tested samples that were stored in the fridge (hence no freeze-thaw cycles) for several weeks and they worked the same.
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* One way to save time would be to re-use standard samples you had prepared previously.   
 +
* Amanda smith has tested samples that were stored in the fridge (hence no freeze-thaw cycles) for several weeks and they worked the same.  Frances freezes them between uses, and re-uses them as long as they aren't more than a few weeks old.
==Contact==
==Contact==
Page set up by [[User:Janet B. Matsen|Janet]] 5/2013
Page set up by [[User:Janet B. Matsen|Janet]] 5/2013

Current revision

Back to Protocols

Contents

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
  • Use the buffer your samples are dissolved in for the diluent.

Materials

  • BCA reagent A
  • BCA reagent B
  • 96 well plate
    • Use BD Falcon plate (VWR item # 29442-070) that is 100% clear and has flat bottoms. The list price is ~$1.75/plate. You don't need sterile plates or plates that are treated for tissue culture; both are more expensive. Polystyrine works well, vinyl may be sensitive to more chemicals like phenol/chloroform.
    • They are kept above the old GC
  • Microcentrifuge tubes
  • Microcentrifuge tube rack
  • Microcentrifuge
  • BSA stock (2 µg/ µl)
  • Pipette
  • Pipette tips
  • Plate reader. Can be either the Tecan (dark colored) or the Molecular devices (white)

Tips

  • Don't wait a whole 30 min before measuring the concentrations in the plate reader. The standards show a linear relationship within 5-10 min. Ceci always reads the plate within 5-10 min.
  • Either plate reader can be used. The Tecan has a 540nm filter for OD measurements.

Procedure

  1. Turn on the plate reader and set it to 562nm & 37oC
    1.  ?? Do you need to turn on the plate reader and warm up the lamp??
  2. 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.
  3. Prepare Standards ( 2 fold dilutions): 2 µg/ µl, 1 µg/ µl, 0.5 µg/ µl, 0.25 µg/ µl, 0.125 µg/ µl
    1. You need at least 1mL of the full-concentration sample (A) to do the dilutions below, so weigh at least 2ug in an eppendorf tube.
    2. Preparing BSA standards for BCA total protein assay
      Preparing BSA standards for BCA total protein assay
  4. 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
      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)
  5. 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

Exporting from SoftMax Pro Software

  • There isn't a way to export excel-like formats. This is very sad!
  • If you want end point data:
    • Change the view option to have:
      • Change the view to show the curve and the maximum value for each well like this:
Image:Export view for Softmax Pro.jpg
Export view for Softmax Pro
      • Make sure that the max value on the curves is well above your maximum absorbance. If only the bottom half of the curve is showing, it the summary number will be the highest number that is within your plot bounds.
      • Save a PDF
      • Type these numbers into excel.
  • If you want kinetic data:
    • You can get a similar view to that above, but displaying v_max.

Re-using standards

  • One way to save time would be to re-use standard samples you had prepared previously.
  • Amanda smith has tested samples that were stored in the fridge (hence no freeze-thaw cycles) for several weeks and they worked the same. Frances freezes them between uses, and re-uses them as long as they aren't more than a few weeks old.

Contact

Page set up by Janet 5/2013

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