I am working in the area of EMT in Cancer cells. I have to perform western blot of an important marker of EMT i.e. Zeb1. The problem is i have tried several times but i am unable to get western blot bands for Zeb1 in my cells. I am working on MDA-MB-231 cells, they have been shown to have good expression of Zeb1 in control lanes in various papers. I have tried every standard troubleshooting method that is possible like increasing transfer time to 70mA for 3 hours and using all fresh buffers with appropriate pH and all. i am using a fresh antibody from CST which was purchased 2 months earlier, so i dont think its an antibody issue. Please help
Hi Kushal,
I would think that a 6-8% gel and an overnight wetblot transfer should work just fine. Though 70mA for 3 hours should be enough as well I assume. Is your marker transferred or have you check with a poinceau staining if your higher MW proteins are transferred? Maybe increase the antibody dilution and perform the primary incubation overnight as well.
Cheers,
A
A 200 kDa protein should get transferred nicely even from 10% gel (40V overnight). Check if your marker bands around 200 kDa or higher are transferred. If yes but still didn't get Zeb1 in WB, make sure your ECL kit works for other antibodies. If ECL reagent is ok but yet Zeb1 didn't show-up, then it could be the lysis issue. Include KCL, NP-40 and TritonX100 in lysis buffer to extract nuclear Zeb1. You may follow the lysis buffer from any published paper that shows Zeb1 in this cell line.
Use a 6% gel for first. Second, reduce percentage of Methanol of 5% (if using PVDF) or nothing (if using nitrocellulose) in your transfer buffer. Good luck!
Try Protein lysate of other cells as control where it is reported, just to make sure your antibody is good. Time and current are fine. If still the problem persist than change the antibody.
A 200 kDa protein can be easily transferred with semi-dry system (1.5 mA per cm2, 45 min with a Bio-Rad semi-dry module). Your transfer buffer should be added 0.05% SDS and reduced Methanol (10% is fine). The iBlot2 system with gradient gels also works well.
I think your western protocol should be fine. However, check whether your antibody is working fine. Sometime cell signaling recommend use 5%BSA for 1ab incubation. Use additional positive control to make sure that lights up at least. Also check your cell line is good.
In wet transfer system, tansfering 200 kDa protein might be difficult. After transfer you can stain your membrane with Ponceau S Red to evaluate the transfer efficiency. if the problem is in transfer so As Katsuaki has mentioned, you should reduce methanol percentage and add some SDS (0.1%) to push proteins to membrane by charging them negatively. All the best :)
All great recommendations so far and I would also suggest using 6% gel for proteins separation, a longer transfer at 4C with buffer containing 0.1% SDS, and confirmation of a successful transfer using Ponceau S or Amido black staining before moving to blocking step. If using HRP-ECL detection, a longer exposure might also be helpful in resolving the issue. Importantly, you may add a positive control from lysates of a cell line which has more of mesenchymal character.
We at Novus have compiled a nice FAQs page on EMT & Stemness. I am sure you will find it useful for your experiments: http://www.novusbio.com/support/emt-and-stemness-faqs
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