[Histonet] i need some technical help!

[Vinnie Della Speranza]

I am sharing information taken from Fluorescent Protein Tracing by R.C Nairn, Livingstone Ltd 3rd ed. 1969. This information is also cited in Principles and Techniques in Diagnostic Histopathology, JM Elias, Noyes Publications, 1982

Liver absorption is frequently employed to purify antibodies simply because  it is a large celullar organ.

liver powder from the species you are studying (in this case Sheep) should be used. You will need to find a source for sheep liver powder. if you cannot locate a source but have access to sheep liver, contact me and I will provide you with the information to prepare your own.

for absorption purposes, 100 mg of sheep liver powder is mixed with 1 ml of the original serum used (your primary antibody). the  proportions are not critical and rough measurement is quite satisfactory.
the mixture is shaken at room temperature for 2 hours at a speed that avoids frothing after which the mixture is centrifuged at 10,000g for 15 minutes and the supernatant is removed

there is no harm in doing the absorption however it isn't clear from your message whether this will solve your background staining problem. can we assume that you are achieving the desired specific staining with the primary antibody and simply wish to clean up the background?

how did you arrive at the correct dilution of your primary antibody ?? is the primary 'home-grown' or purchased commercially ??

please let us know the outcome of the absorption


Vinnie Della Speranza
Manager for Anatomic Pathology Services
Medical University of South Carolina
165 Ashley Avenue  Suite 309
Charleston, SC 29425
Ph: 843-792-6353
fax: 843-792-8974

>>> Mr srinivas sv <drsvsrini <@t> yahoo.com> 04/28/05 05:34PM >>>

Hi,

Does anyone have a protocol or reference for cleaning up polyclonals
with acetone-extracted liver powder? 

I am trying to look at the expression of steroidogenic enzymes in the sheep ovary. My sections are cryosections. All my antibodies are polyclonal antibodies. My problem is that there is color development in both positive and negative controls. I am using normal rabbit sera (from SIGMA) for the negative control as my primary antibodies are raised in rabbit. I am using Normal goat sera and 1% BSA for blocking nonspecific antigens (Serum Blocking) as my secondary antibody is raised in Goat. I am also using 1% hydrogen peroxide for blocking endogenous peroxides. Fed up with this, i now want to try using acetone-extracted liver powder as an adsorbent. Can anyone explain me the use of acetone-extracted liver powder and protocol for using it for my experiment. 

I am trying this from past 6 months, without any positive result. So, anyone please troubleshoot my problem and suggest any alteration in my protocol. 

Please feel free to ask for any clarifications. 

Thanks... 

Regards, 

Srinivas




Srinivas Seekallu 
PhD student
Dept of Vet Biomed.Sci.
Western College of Veterinary Medicine,
University of Saskatchewan.
Saskatoon, Canada.

Ph: 306-477-0849 (Home)
    306-966-7373 (Lab)
    306-966-7382 (Office)
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