antibody storage - how to store? (Jul/15/2008 )
Hi all,
I have a problem of antibody storage
I purified my antibodies using Protein G column
loading and washing buffer is 20mM Sodium phosphate buffer (pH 7.0)
elution buffer is 100mM Glycine pH 2 and 3
antibodies are neutralized in 100ul of 1M Tris pH 8.0 for 1ml fractions by collecting the eluates directly into tris containing tubes
the eluates with peak fractions were pooled and dialyzed against 20mM Tris pH 8.0, 40mM NaCl, 1mM EDTA and 20% glycerol
after dialysis, i aliquoted the antibodies and stored at -20 after snap freezing in liquid nitrogen
i observed that there is loss of activity of antibody drastically
after this i tried storing the antibody without any freezing directly at 4C
even after this the activity is lost within 15days
can someone pease suggest me what is the thing i am doing wrong in the storage or even purification procedure
awaiting for your valuable suggestions
Leelaram
I have a problem of antibody storage
I purified my antibodies using Protein G column
loading and washing buffer is 20mM Sodium phosphate buffer (pH 7.0)
elution buffer is 100mM Glycine pH 2 and 3
antibodies are neutralized in 100ul of 1M Tris pH 8.0 for 1ml fractions by collecting the eluates directly into tris containing tubes
the eluates with peak fractions were pooled and dialyzed against 20mM Tris pH 8.0, 40mM NaCl, 1mM EDTA and 20% glycerol
after dialysis, i aliquoted the antibodies and stored at -20 after snap freezing in liquid nitrogen
i observed that there is loss of activity of antibody drastically
after this i tried storing the antibody without any freezing directly at 4C
even after this the activity is lost within 15days
can someone pease suggest me what is the thing i am doing wrong in the storage or even purification procedure
awaiting for your valuable suggestions
Leelaram
Antibody storage:
Add Albumin &/or Glycerol if you plan to keep them at -20'C.
Add azide if you plan to keep them at 4'C.
For more: http://search.vadlo.com/b/q?keys=antibody+...mp;sn=158621799
..
If you can add BSA as a bulking agent this may help. I would suspect the drastic pH change had an effect on the ab. Hopefully, after collection, you immediately adjusted the pH. I would dialyze against Hepes or other buffer that is used or would be used in tissue culture to mimick the environment the ab is commonly in.
Also, make sure you have a sufficient concentration 1 mg/ml would be ideal.
Azide or thimerosal for liquid storage.
Good luck.
Hi,
thank you for your valuable suggestions
i would try adding BSA but how much percentage
also will dialysis against PBS help
awaiting your replies
Leelaram
If BSA will not interfere with the final use of your ab then 0.05% should be fine. I would strongly suggest HEPES for storage as it can be used for tissue culture.
Hi all of you,
thank you all for your valubale suggestions
I dialyzed my antibody with the procedure mentioned earlier in this topic against PBS
and stored in 2% BSA. I observed that the antibody is pretty stable. and i stored in 4C.
also i want to do some molecular biology experiments . hope this has solved the problem
now i want to transfer them to -20C. so can i place the vials directly into -20 or shall i have to snap freeze in liquid nitrogen
awaiting your replies
Leelaram
thank you all for your valubale suggestions
I dialyzed my antibody with the procedure mentioned earlier in this topic against PBS
and stored in 2% BSA. I observed that the antibody is pretty stable. and i stored in 4C.
also i want to do some molecular biology experiments . hope this has solved the problem
now i want to transfer them to -20C. so can i place the vials directly into -20 or shall i have to snap freeze in liquid nitrogen
awaiting your replies
Leelaram
no need to snap freeze. we just put it into a -20C freezer.
Hi all.
thank you for your suggestions