RE: smooth muscle cell actin

<< Previous Message | Next Message >> (Peter van de Plas)
Date:Thu, 03 Jun 1999 23:20:45 +0200
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Silvia wrote:
Does anyone out there have experience with non-specific nuclear
staining artifact?
I've been having problem with immunostaining smooth muscle alpha
actin (Sigma A-2547) on cultured human smooth muscle cells.
I did a few optimizing test on the antibody. It was working fine at
1:400, stained the cytoplasm nicely and nice & blue counter stained
nuclei with haematoxylin.  The antibody's been frozen in aliquotes.
Three four months later I use it again, it stains all nuclei dark brown. All
Any suggestion?
Dear Silvia:
Clustering of your primary antibody seems to me the most obvious reason for
your background problem (I presume your negative control is oke). Possible
remedies are:
- (ultra)centrifugation of the diluted antibody. Normally I use  an
eppendorf centrifuge at max speed for 10 min.
- increase of pH of the antibody solution to 8-9.
- addition of sodium chloride (0.5-1.5M).
To prevent clustering of future batches: add glycerol in a 1:1 ratio before
Hope this is of help.

Peter van de Plas
Costerweg 5
6702 AA Wageningen
The Netherlands
phone: (31)-317-497676
fax: (31)-317-415955

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