[Histonet] Hard Antibodies! - long response

[Liz Chlipala]

Emily

When you purchase antibodies from vendors they "hopefully" have gone =
through a process in which the antibody has been purified, this =
information is on the specification sheet under antibody form, there are =
several forms of antibodies, such as:

Affinity Purified IgG
Stabilized Antisera
Ascites Fluid
Cell Culture Supernatant
Whole Antiserum
IgG Fraction=20

Most vendors will tell you what the antibody form is and what the =
purification method was.  Purification is the removal of unwanted =
immunoglobulins or IgG's that do not react with the protein of interest. =
 Vendors should also  determine the specificity and sensitivity of the =
antibody which can be done through a series of immunochemical techniques =
including but not limited to immunoelectrophoresis, westerns blots, =
double diffusion, rocket immunoelectrophoresis and ELISA, this =
information is also sometimes listed on the specification sheet.  If not =
you can always call the vendor.  In addition to these techniques they =
could also test the antibody on a wide range of positive and negative =
tissues with different staining protocols.  But ultimately it=92s the =
user=92s responsibility to ensure the quality of the primary antibody =
prior to use, this is what we do with our titers studies and validation =
protocols.  =20

Antibodies that are so called "in house" antibodies are or are not =
purified.  Unpurified antibodies can consist of a wide variety of =
different immunoglobulins some that are directed against the protein of =
interest and some that are not.  It is very difficult to deal with these =
types of unpurified antibodies.  I have not had the opportunity to work =
with many, but overall I would have to say that they don't work that =
well.  One thing that needs to be considered is what is the protein =
concentration of the antibody of interest?  How can you determine that =
when you have other IgG's in the mix, if you do not know the protein =
concentration or form of your antibody how can you possibly set up the =
appropriate negative control?  When getting an "in house" antibody in =
for evaluation I would definitely ask the following questions:  Has it =
been purifed? what form is it?  Whats the protein concentration? And if =
any testing has been performed regarding to specificity?=20

Regardless of whether you are dealing with a commerically available =
antibody or an "in house" preparation careful review of the =
specification sheet along with current literature is necessary.  You =
need to know as much as possible about what you are working with inorder =
to be able to evalute the results of the titer studies. =20

Just my two cents, whatever its worth.  I'm stepping off the soap box =
now.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
P.O. Box 18592
Boulder, CO 80308
phone (303) 735-5001
fax (303) 735-3540
liz <@t> premierlab.com
www.premierlab.com
=20
Ship to Address:
=20
Premier Laboratory, LLC
University of Colorado at Boulder
MCDB, Room A3B40
Boulder, CO 80309

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu =
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Emily =
Sours
Sent: Saturday, June 16, 2007 2:46 AM
To: James L Burchette
Cc: Histonet <@t> lists.utsouthwestern.edu; =
histonet-bounces <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] Hard Antibodies!

regarding your hard-to-process antibodies--are they manufactured by a =
company, or has an individual made them? does this make a difference?
in response to my own question, the antibodies we receive from =
Developmental Studies Hybridoma Bank (DSHB, University of Iowa), =
Molecular Probes, and Rockland Immuno work very well.  our problem is =
with antibodies from individual labs.
could this be a problem with shipping? the antibodies we receive from =
individual labs are not on dry ice, but a PI told me that since human =
antibodies can survive at high temperatures, shipped antibodies can =
survive it as well.*

Emily
*all of this does not apply to Chemicon antibodies, which suck.
--
penguins, spoons, and you--what's life like among the flightless?
http://www.getafirstlife.com/

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