affinity purification of antibodies - what is the best buffer to elute (Aug/30/2007 )
We are planning to get our antisera affinity purified. I was wondering if anyone else had any experience with that!!
I had two doubts: -
1). what is a good buffer to elute the antiibody out in? PBS? anything else?
2). Also, does anyone know what stabilizing agent (cryo protectant) is added to say commercially available antibodies. We have the option of 50% glycerol, maybe BSA!!
I really appreciate any suggestions.
for your point 2 : i would redirect you to this topic.
Please find a quote
question : What is the best way to store antibodies for extended periods of time?
Should they be stored at -80 C or at -20 C. I have heard it is better to
store them in glycerol (1:1) to perserve them for a longer period of time.
1. Purified monoclonal and polyclonal antibodies-store 1:1 in glycerol at-70C
2. Polyclonal antiosera , -20C is fine (the albumin provides protection).
3. Conjugated Abs (eg HRP or AP..tricky, if you made them , store them 1:1 in glycerol, if you bought them, follow manufacturers instructions.
answer 2 :
Several antibody storage strategies are documented and, in most cases, will work with most antibodies. One should determine, on a small scale, whether a particular antibody has a stability issue. For example, I recently discovered that an epitope affinity-purified polyclonal IgG stored in the presence of BSA fails to bind antigen after a single freeze-thaw cycle yet the total, unpurified IgG fraction immunoblots perfectly after numerous freeze-thaw cycles. Here are some storage conditions I have used with good results.
1. Sterile-filtered crude antisera with 0.05% Na Azide at 4 C for 1-2 years; -80 C almost indefinately.
2. Ammonium sulfate ppt. IgG slurry at -20/-80 C almost indefinately.
3. Crude ascites or purified IgG from ascites at -20 C/-80 C almost indefinately.
4. Affinity-purified IgG with at least 1 mg/ml BSA at -20 C/-80 C almost indefinately.
5. Antibody 'working stock' with 50% glycerol at -20 C for 6 mo-2 years.
I hope this is useful to you. Please contact me with additional questions.
Phillip E. Schwartz
Antibody Partnership Project Manager
Affinity BioReagents, Inc.
14818 W. 6th Ave., Suite 13A
Golden, CO 80401
email: schwartz at bioreagents.com
Thanks a lot for the suggestions Fred_33.