Fong:Bradford protein assay

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Revision as of 12:21, 20 March 2009 by Christopher M Gowen (Talk | contribs)
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Contents

Overview

This protocol is a relatively simple way to quantify total protein based on the Bradford method, in which Coomassie dye undergoes an absorbance shift from red to blue in the presence of protein. It is a subjective assay, dependent on amino acid content of the sample, so it is important to use controls and to interpret the results carefully. For example, protein can be quantified in units of mg equivalent bovine serum albumin.

This assay is commonly used to monitor cell growth when culturing on insoluble substrates such as cellulose, so it is useful to correlate this data to dry cell weight, optical density (on cellobiose or another soluble substrate), or cell counts. This protocol is therefore divided into two parts: first, the preparation of cell suspensions to remove interfering cellulose and obtain a solution of cellular protein, and second, the actual assay protocol.


Materials

  • 0.9% m/v NaCl
  • 0.2 M NaOH
  • Protein assay dye 1x (BioRad)
  • Clear round-bottom Nalgene centrifuge tubes
  • 2ml microcentrifuge tubes
  • 96 well plate
  • Heating block
  • Centrifuge capable of spinning 10ml samples at 8000g, located in 407
  • Plate reader

Procedure

Preparation of protein solution from cell suspension

  1. Turn on heating block to 100°C. Remove a 10ml sample from the well-mixed cell suspension, and centrifuge it at 8,000g for 15 minutes. Discard supernatant.
  2. Wash pellets with 0.9% m/v NaCl, spin again, and resuspend in 2ml 0.2 M NaOH..
  3. Incubate for 10min in the heating block at 100°C.
  4. After cooling, centrifuge at 8,000g for 15 min. Collect supernatant for Bradford assay.

Protein Assay (Bradford)

  1. In 96 well plate, carefully pipette 100μL protein assay dye 1x (BioRad) in each well.
  2. Add 20μL sample, mix well by pipette.
  3. Incubate at room temperature ~5 min. Read A595.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

  • Christopher M Gowen 09:55, 19 March 2009 (EDT)The dye and sample mixture tends to be detergenty, so bubbles form really easily and can interfere with absorbance reading. Don’t push out the last bit of sample from the pipette because this exacerbates bubble formation.
  • Chris 13:21, 20 March 2009 (EDT) This can also be done with smaller volumes down to ~2ml. It may be necessary to resuspend washed pellets in a smaller volume of NaOH to get nice clear readings on the plate reader.

References

  1. Sparling R, Islam R, Cicek N, Carere C, Chow H, and Levin DB. . pmid:16917525. PubMed HubMed [Sparling2006]
    cell prep method was adapted from here

Contact

Chris

or instead, discuss this protocol.

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