[Protein-analysis] Re: (no subject)

[allisonh]

You could try probing in the opposite order: Syk then 4G10


Nichol Holodick wrote:
> 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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