Hi All,
Can anybody suggest what are the important points to consider when transferring high molecular weight proteins for western blot analysis. (Apart from low % of PAGE, less methanol and addition of SDS in transfer buffer).
I am facing problems in detecting a 290 KD protein by WB. So I presume the transfer is the key step where I should work on.
Anyway, if anybody has any special suggestion for this, that would be great.
Thanks.
You could perform a quick dotblot to be sure you are using the right amount of detection antibody for your protein. You can do this by dripping some of your solutoin with protein directly on your membrane, let it dry and start your immunedetection. Also, do not cut off your stacking gel and keep it intact throughout the procedure. Additionally, you could stain an entire gel (+stacking) with coomassie blue to guestimate where your protein is going after electrophoresis.
You could perform a quick dotblot to be sure you are using the right amount of detection antibody for your protein. You can do this by dripping some of your solutoin with protein directly on your membrane, let it dry and start your immunedetection. Also, do not cut off your stacking gel and keep it intact throughout the procedure. Additionally, you could stain an entire gel (+stacking) with coomassie blue to guestimate where your protein is going after electrophoresis.
Indrajit Bhai,
What % gel are you using? As per my experience, a 3.5 or 4 % gel with all other mentioned tricks, you should be able to detect your protein of interest.
I have always used this for APC (full length) (310 KDa).
Let me know.
Good luck!!!
Wet transfer is the best for large proteins (i used to do wet transfer for at least 2 hours). Also, I got much better results when I used the tris-acetate 3-8% gels (Thermo Fisher Scientific) - highly recommended. Coomassie blue staining to see what has been transferred, as mentioned above, is a good suggestion. Good luck!
Even with proteins around 300 kDa (mTOR comes at 289 kDa), we are using 10% gels and 50 min blottime with 100 V in a tank blot system which is working fine. For higher molecular weight proteins, we were using bis-tris gels and blotting over night at 10V (something like 16 h) and up to 460-480 kDa was not an issue.
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology