ANSWER: |
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Thank you for that information.
I placed the order for you to receive ab29475, rat brain wholecell lysate to use as a positive control with ab54421. Unfortunately it is out of stock at the moment and we have an expected delivery date of 26th January. When we receive the product back in stock, we will ship to you as soon as possible. I am sorry about this inconvenience.
I am unsure as to the exact method that the cell lysate was produced, however it should contain your protein of interest.
If you are still having problems with ab54421 after you receive the positive control, please let me know. |
Thanks for your prompt reply! Attached is the WB image. Please see my
answers to your questions below.
>
> I am sorry that you are having problems with ab54421 in western blotting. I was wondering if you provide me with some more information about how you prepared your sample as Alpha 1-subunits of voltage-gated Ca2+ channels are highly sensitive to proteases. All procedures involving a full-length protein should be performed at 4°C and include protease inhibitors.
The mouse brain lysate was prepared by adding 10 fold volume of
ice-cold lysis buffer (50mM Tris pH7.6, 150mM NaCl, 5%glycerol, 1%
NP40, 1mM EDTA, plus freshly added protease inhibitors including 5mM
Benzamidine, 5mM Antipain, 5mM Leupeptin and 1mM PMSF.) to -80C frozen
whole brain, and dounced 40 times on ice, followed by rotary mixing
1hr at 4C, and clarified by centrifugation at 4C, aliquoted, snap
freozen, stored at -80C and thawed before each use. I can detect all
other tested large transmembrane proteins from this lysate by WB, with
molecular weight ranging from 100kd to 250kd.
>
> Also could send the western blotting protocol that you have been using, including primary antibody concentrations used, incubation times, blocking buffers and also for the transfer buffer recipe that you used. Having this information will help try narrow down the reasons why you are not seeing the correct size band.
Clarified lysate was mixed 1:1 with 2X loading buffer, and boiled
12min. I loaded ˜15ug total protein to a 10% PAGE gel, and transferred
to PVDF membrane by conventional wet method at 80V for 90min. Transfer
buffer is the standard Tris-glycine buffer with 20% methanol - no
problems for another 250kd membrane protein, and transfer seems
complete based on the 250kd band from a kaleidoscope marker (BioRAD).
The membrane was blocked in 5% fat free milk in TBST (20mM Tris pH7.6,
140mM NaCl, 0.1% Tween20) at RT 1hr, then rinsed and incubated with
your anti-Cav1.3 antibody, 1/1000 dilution in 2% BSA in TBST, for
overnight at 4C. The membrane was washed five times with TBST and
incubated with HRP-conjugated goat-anti-rabbit secondary antibody
1/10000 dilution in 2% BSA in TBST for 1hr at RT. After being washed 5
times in TBST, the membrane was developed by regular ECL (GE
healthcare).
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Thank you for reply and providing your protocol information.
I have to admit I am little perplexed as why you are getting such a clean band at 150kDa but not at 250kDa, your sample preparation and western blot protocol are spot on, the only suggestion I could make it to increase the concentration of protease inhibitors used, since it is known that Alpha 1-subunits of voltage-gated Ca2+ channels are highly sensitive to proteases.
There are 2 options that we can explore to see what the issue is:
1 - I can send you an aliquot of rat brain lysate that you can run as a positive control. I know your sample is good, but the WB image on the website is of rat tissue and although I cannot find any reference to different species having different isoforms, it could be worth a try.
2 - I could send you another CaV1.3 antibody to try out (possible ab85491).
Please let me know how you would like to proceed and I will be happy to try and resolve this issue for you. |
Hi, I used your anti-Cav1.3 antibody (ab54421) today for western blot, and found that it did not give me expected band. It is supposed to detect cav1.3 about 250kd, but it detected a product about 150kd. I am blotting clarified whole mouse brain lysate solubilized by 1% NP40. Would you please suggest how to troubleshoot? Thanks! |
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Thank you for contacting Abcam.
I am sorry that you are having problems with ab54421 in western blotting. I was wondering if you provide me with some more information about how you prepared your sample as Alpha 1-subunits of voltage-gated Ca2+ channels are highly sensitive to proteases. All procedures involving a full-length protein should be performed at 4°C and include protease inhibitors.
Also could send the western blotting protocol that you have been using, including primary antibody concentrations used, incubation times, blocking buffers and also for the transfer buffer recipe that you used. Having this information will help try narrow down the reasons why you are not seeing the correct size band.
The antibody is covered under our Abpromise for six months and is guaranteed to work in western blot on mouse samples . If we cannot resolve the issue you are having with the antibody then I would be happy to either send a replacement antibody or to process a refund.
I look forward to your reply and helping resolve this issue. |
