Protein assays and detergents

cthomas at science.adelaide.edu.au cthomas at science.adelaide.edu.au
Sun Jun 27 19:31:26 EST 1993


Thanks to all those who responded to my request for help concerning a
method for determining protein concentrations in the presence of detergents
such as SDS, Triton etc.  

Several people requested that I post a summary of responses to my query. 
The remainder of this posting lists most of these responses.

Connor Thomas
Dept Microbiology and Immunology
University of Adelaide
cthomas at microb.adelaide.edu.au

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From:   suhy at casbah.acns.nwu.edu

Read your posting on the mobionet.  We have used an assay in our lab here at
Northwestern University for protein concentration.  It is a product sold by
Bio-Rad here in the USA.  Their address is:

Bio-Rad Laboratories
2000 Alfred Nobel Dr.
Hercules, California 94547

Telephone: 1-800-424-6723
Fax: 1-800-879-2289
Telex: 71-3720184

The assay system is fairly simple and straight forward and is compatible
with a wide variety of detergents including 10% SDS, 1% triton x-100, and 2%
NP-40.   The kit is also fairly inexpensive - ~ $100 US dollars.  Hope this
information helps you out.

Dave Suhy
Department of Biochemistry,
Molecular Biology, and Cell Biology
Northwestern University
USA

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From: Basavaraju Shankarappa <bsh at med.pitt.edu>

Check out the BCA protein assay system from Pierce.  I have had very
good experience with this reagent.  It is compatible with upto
1% NP-40 (micro version), 1% SDS etc.,.
Raj Shankarappa
bsh at med.pitt.edu

----------------------------------------------------------------------------------------
From: GR8BN at CC.USU.EDU

Dear Dr
There is a kit suppliedddby Pierce Chemical Co. Rockford, Illinois 61105, USA
can be used for determining the concentration of protein without interference
by Tx-100 and SDS. Good luck !

----------------------------------------------------------------------------------------
From: shaun at jason.decnet.relay.the.net
Message-Id: <01GZN1KZCK3M000U4U at nic.the.net>

     The Bradford is notoriously detergent sensitive (see BioTechniques 4,
130-136 (1986), or Anal. Biochem. 178, 263-268 (1989).  Instead, we use the
BioRad BCA (bicinchoninic acid assay).  It is a modification of the Biuret,
and as such is quite linear with protein, fairly independent of the
particular protein, and remarkably insensitive to a wide variety of agents,
including most detergents.  For reference, see Anal. Biochem. 150, 76-85
(1985) for the primary method, and Anal. Biochem. 180, 136-139 (1989) for
an investigation into interfering substances.  Hope this helps.  Cheers,
   Shaun

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From: Andy Phillips <PHILLIPSA at UK.AC.AFRC.LARS>

For my Ph.D., I used an adaptation of Lowry which removed detergents
by precipitation with TCA (this was 1976, by the way). You might
be able to adapt this for Bardford. The ref is Bensadoun and Weinstein
(1976) Anal Biochem 70, 241-250

Andy Phillips

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From: pas at aretha.jax.org (Peter Schweitzer)
Organization: The Jackson Laboratory, ME, USA

You might want to try the bicinchoninic acid assay (BCA), which I have been
using for some time.  It works quite well in the presence of 1% NP-40, and
it is easily adaptable to small samples (5 microliters).  I also read the
samples using an ELISA plate and automated reader equipped with a 570 nm
filter (optimal is 562). The sensitivity in my hands is 20 micrograms/ml.

One reference is: Smith, P. K. et al,. Analytical Biochemistry (1985)
150:76-85.

A kit available from Pierce works great, and individual reagents are
available from Sigma (hopefully you can find a source down under).

Good Luck

PAS

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From: mark at biosci.mbp.missouri.edu (Mark Horn)

    I have work in protein chemistry for quite a while and I have found
that using a chloroform-methanol ppt. method best for determining protein conc.
in detergent containing solutions. the reference for this is:

Wessel and Flugge Analytical Biochemistry 138: 141-143.

mark

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From: Hsing-Tsu Lai <BMHTLAI%TWNAS886.BITNET at pucc.Princeton.EDU>

Hi!
BioRad sells a assay kit called DC (Detergent-Compatible) Protein Assay.
Therefore, you might get a reference from its representatives.
Good Luck!

bmhtlai at ccvax.as.edu.tw

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From: "Richard P. Grant" <rpgrant at molbiol.ox.ac.uk>

BioRad do a Bradford-based kit which sounds tailor made to your needs :)

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From: HARVEY at UK.AC.AFRC.JII

Have a look at :
Schaffner and Weissman , Anal Biochem 56, 502-514 (1973)

see J biol Chem 266, 9153-9160 (1991) where technique was used.
regards

David Lonsdale



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