RE: [Histonet] Hard Antibodies! - long response

From:"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 

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 itís the userís responsibility to ensure the quality of the primary antibody prior to use, this is what we do with our titers studies and validation protocols.   

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? 

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.  

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@premierlab.com
www.premierlab.com
 
Ship to Address:
 
Premier Laboratory, LLC
University of Colorado at Boulder
MCDB, Room A3B40
Boulder, CO 80309

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