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Thank you for your suggestions. In reviewing my previous blots, I
realized that all the ones I had run with the commercial cell lysate
had been stained with Amido Black, which made them unsuitable for
stripping and re-probing. I consequently ran another gel, loading
15 ug of ab30151 as well as the lysate for the system I'm trying
to study. I ran my transfer similar to before (75 V for 4 hours),
using 5% MeOH and 0.5% SDS in my transfer buffer, similar to your
suggestion. After transfer, I can see my MW marker (other company) fully transferred. I then pre-blocked with 3% BSA before
incubating with my primary antibody (ab65166) in 3% BSA overnight
at 4 oC. Finally, I incubated with the secondary antibody in 3%
BSA for 1 hour at room temperature and then did 3 10 minutes washes
with TBS-Tween before a final wash with TBS.
Upon visualization with my ECL+ kit, I only saw very high background
at moderate exposure times (1 minute) and couldn't see any signal
above background at very short exposure times (5-10 seconds). In
hindsight, I believe I should have incubated with the secondary
antibody in 10% milk.
I tried stripping the blot using the mild stripping protocol on
your website but still saw an almost equally strong background
upon visualization. At this point, I assume I must still have a
lot of secondary antibody non-specifically bound to the membrane,
so my next step is to try the harsh conditions.
Even though I see my full MW ladder transferred, I could definitely
believe that my transfer is still sub-optimal. I would like to try
alternative transfer conditions, but I have very little of my
remaining lot of ab30151 left. Would it be possible for me to obtain
another lot of this lysate at a discounted price? Alternatively, is
there another item in your catalog that you would recommend as a
positive control for the primary antibody that I could pay full price
Asked on May 15 2012
Thank you for your reply.
I have a few additional suggestions. I am not sure if my colleague had mentioned those to you:
1) You can check the transfer with Ponceau S (which is a reversible membrane stain) to be sure that you transfered the proteins. This will give you also an additonal quality control for the transfer. My feeling is that transferingat 75V for 4 h could be too long. if the gel can withstand a higher voltage, I'd suggest to try 100V for an 1 h at 4 degree with stirring.
2) Further, I'd suggest to use 10% MeOH and reduce the amount of SDS to 0.1% in the transfer buffer. You want to have some SDS to prevent hydrophobic proteins (like Nav1.6) from precipitating in the gel during the transfer, but 0.5% seems a bit high.
3) Are you blotting for any other proteins, such as actin or tubulin as loading controls? This would be another way to check if the WB protocol in general works and if the mouse brain lysate you have is OK.
4) How are your samples lysed? I would recommend using RIPA buffer since the Nav1.6 protein is very hydrophobic.
I also checked online and found the following link for WB with membrane proteins:
Our WB protocol guide can be found here: https://www.abcam.com/index.html?pageconfig=resource&rid=11375and I have it also attached to this email.
To answer your question about the lysate:
I'd be happy to send you another vial of ab30151 free of charge. Alternatively, we also have ab7188 (Brain (Mouse) Tissue Lysate - normal tissue, 0 days old) [https://www.abcam.com/Brain-Mouse-Tissue-Lysate-normal-tissue-0-days-old-ab7188.html] in the catalog, but I am not certain if newborn mice express this protein strongly.
I can send you 1 vial of either one or the other free of charge. Please let me know which one you would prefer. Please let me know your order or PO number for the lysate.
I look forward to hear back from you and assist you further.
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Answered on May 15 2012