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Old 10-08-2007, 07:03 PM
Nichol Holodick
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Hello Phil,

I have seen you post online many times offering help to people who have questions about stripping membranes. I was wondering if you have any advise for my problem. I performed an IP with Syk and then probed with 4G10. Upon stripping
(Bio-Rad Restore Stripping buffer for 30 minutes and then 30 minutes
washing in TBS-T) and re-probing for total Syk protein pulled down I
see white bands where Syk should be. Thinking I just had too much
primary antibody I stripped again (same as first strip) and I reduced
the dilution form 1:1000 to 1:3000, but again the white bands.

Then
I thought, maybe since the 4G10 antibody is a monoclonal antibody and
has very high affinity I may not be completely stripping the 4G10 off
and therefore what I am seeing is the 4G10 blocking the ability of the
Syk antibody to bind. So I stripped again under the following
conditions: 10mM Tris pH 6.7, 2% SDS, 100mM 2-ME at 56C for 40 minutes.
This seemed to help a bit more. This time I could see the Syk band
where it should be but in a few lands where the IP showed a stronger
signal I still see white bands!!

I really need these loading
controls, otherwise many very expensive experiments are not very
useful, as you all know. I am just not sure what to do next. I am
planning on stripping again with 0.2M NaOH but am not sure this will
help or hurt? Any suggestions would be really appreciated!

Thank you for your time and help.
Nichol
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Old 10-09-2007, 10:28 PM
allisonh
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You could try probing in the opposite order: Syk then 4G10


Nichol Holodick wrote:
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