Hi, recently I did a western-blot, but something went wrong during the transfer step. I did my transfer at 120V for 1.5h on a 12% gel (1.5mm) to a PVDF membrane, however, now I'm not able to see my ladder anymore.... Dit my proteins go through the membrane?
I think 120V might be to high for transferring 1.5h long,....
Based upon your voltage listed, you performed a wet transfer, correct? Whenever I do a wet transfer, I transfer at 100 Volts for 30-40 min depending upon the size of the protein I will be looking at with antibodies. I do think that you transferred your gel for too long of a period. I do not think that the voltage was necessarily too high, but the duration certainly was in my opinion.
Other simple questions, you made sure to activate your pvdf membrane with 100 % methanol, correct?
Did you ponceau your blot after the transfer? That would certainly tell you if you lost all of your proteins due to over transfer
Hello Lauren
There are many reasons which I can think about.
1. In the transfer apparatus I hope you have used the right sequence of gel and membrane namely, filter paper-gel-membrane-filter paper.
2. The PVDF membrane must be pre-wet in 100% methanol for 5 minutes and then equilibrated in transfer buffer before use.
3. I hope the sponge or pads and filter papers are presoaked before making the sandwich.
4. All the air bubbles trapped between the layers in the sandwich prepared must be removed with the help of a clean pipette by rolling it over the sandwich in order to ensure good contact between layers.
5. Use 20% methanol in the transfer buffer as methanol helps to remove SDS molecules bound to the protein and facilitates binding of protein to the membrane.
6. Precool transfer buffer and carry out transfer process at 4 degrees celsius either on ice or in the cold room.
7. The voltage can affect transfer. So, use 30-50volts for 1 hour. Increased transfer time could increase the risk of over transfer of protein through the membrane especially for low molecular weight proteins of less than 30kDa when using membrane of large pore size such as 0.45um.
8. For wet transfer the gel side of the cassette holder should face the cathode while membrane side should face the anode.
9. Lastly, check for the transfer by staining the membrane with Ponceau S or the gel with Coomassie Blue.
I hope this is helpful.
Best Wishes.
Try and use some Ponceau Red in that membrane, it will show you if proteins are on the membrane or not,. Sometimes you do not get to see your ladder very well, but you can still have proteins in your membrane. If there is a problem with your Blot, you have already plenty of advices in the other answers, so you can find your problem.
Thank you Brandon A Kemp , Malcolm Nobre , Jorgaq Pata for the advice! According to what you recommend, I think I did everything correctly besides the long transferring time. My supervisor told me to not do the Ponceau staining and to start over. Second time better luck
- Badges
- Science topic
- Similar topics
- Cognitive Science
- Thinking