Anti-Nav1.6 antibody (ab65166)

Thank you for your helpful advice. I used Ponceau S on my most
recent blot and was able to visualize transfer of both my MW marker
and proteins in my cell lysate. It's certainly possible, however,
I could be getting over-transfer to some extent. I recently contacted
the lab that wrote the review for ab65166 on your website, and they
said that they performed a 16 h transfer at 20 V. With the new lysate,
my hope is to try both your one hour transfer conditions and the 16 h
transfer conditions and see if I see better transfer with either.
I'd also be open to trying higher MeOH and lower SDS content;
although, the aforementioned group apparently used a basic transfer
buffer with 20% MeOH and no SDS.
I'm not currently blotting for a loading control, but that is
a good suggestion. The best evidence I have for the success of my
WB protocol is that I could visualize Invitrogen's Precision
Plus MW marker after incubation with my secondary antibody.
In terms of lysing my own sample, I used the following reference to
prepare my lysis buffer (http://www.springerprotocols.com/Full/doi/
10.1007/978-1-61779-328-8_23?encCode=U0VOOjMyXzgtODIzLTk3NzE2LTEtODc5
&tokenString=k/NnQ1wdXIf1ihPjyV6QSQ==).
I certainly am open to try other lysis buffers but felt it might
be best to use a formulation that had been used successfully with
these channels before.
I very much appreciate the offer to send me another vial of ab30151.
From some initial reading, it looks like Nav1.6 expression increases
during maturation, so it's perhaps best to stick with the lysate
I'm already using. The order number for the original order is
xxx.
I can't thank you all enough for your help so far. Your team
has been incredibly responsive, and I very much appreciate your
assistance.

ANSWER:

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement with the order number xxx. The estimated delivery date is xxx as the item is currently backordered. I hope this is not causing a big problem. I apologize.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

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
for?

ANSWER:

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:
http://www.sciencedirect.com/science/article/pii/S0003269708006829

Our WB protocol guide can be found here: http://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) [http://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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No signal in western blots

ANSWER:

Thank you for calling and bringing this issue to our attention.

I just wanted to make sure you were loading the correct amount of protein and to make another suggestion regarding the blocking step.

For the brain lysate, ab30151, you will want to load at least 10ug, which is a volume of about 4 microliters.

For the blocking step, you may find that a relatively short block (30 minutes) instead of overnight, followed by overnight incubation with the antibody will help develop the signal, compared to an overnight block followed by a shorter incubation with the primary. These are little modifications, though. I suspect that either the lysate is defective or the transfer was not optimal. Please let me know how your tests go. If they fail, we will be happy to discuss a replacement of either the antibody or the lysate, or both.