weak purified antibody - (Apr/07/2010 )
Dear all,
I purified my protein by using agarose A, the SDS PAGE showed the bands related to my antibody ( light and heavy) and when I used t in immunostaining the staining was very faint !! can anyone give me explenation.
-bluebird-
Have you measured antibody titre in serum and purified antibodies with ELISA? That should give you better comparison.
What are you using for elution - pH or chaotrope? Perhaps you're denaturing your antibodies a bit and that's why you loose activity?
-K.B.-
also, was the antibody made against native or denatured protein?
-mdfenko-
K.B. on Apr 7 2010, 06:56 AM said:
Have you measured antibody titre in serum and purified antibodies with ELISA? That should give you better comparison.
What are you using for elution - pH or chaotrope? Perhaps you're denaturing your antibodies a bit and that's why you loose activity?
What are you using for elution - pH or chaotrope? Perhaps you're denaturing your antibodies a bit and that's why you loose activity?
yes I recived the titer of the antibody t was positive but not high positive, the protocl that I follow is the same described by Sigma for protein A agarose. the elute pH was 3.
I tested my antibody in cell line expressing protein specific for this antibody.
-bluebird-
bluebird on Apr 7 2010, 01:06 PM said:
yes I recived the titer of the antibody t was positive but not high positive, the protocl that I follow is the same described by Sigma for protein A agarose. the elute pH was 3.
I tested my antibody in cell line expressing protein specific for this antibody.
I tested my antibody in cell line expressing protein specific for this antibody.
did you neutralize after elution at pH 3? you don't want to let it sit at low pH for long.
-mdfenko-