Hello everyone,
I am right now facing the issue of air bubbles during the western blot wet transfer method. It is so frustrating that some silly air bubbles mess up the whole results. This is what I do:
I run an 8% SDS-PAGE gel, use bolt & mahoney transfer buffer, use nitrocellulose membrane for protein transfer. The transfer conditions are 25V, 300mA, constant voltage at 4C, with constant stirring. I leave this transfer overnight. At the end of the transfer time, I remove the membrane and wash it with 50ml of distilled water 2x5min. Later, I stain it in Ponceau solution for 30seconds, followed by 2x5min wash with 50ml of distilled water. I take the utmost care to prevent any air bubbles while setting up the sandwich. I also use the teflon roller to roll out any air bubbles which may have been trapped in between the gel and the membrane.
I am so at the dead-end of what other tricks I could employ to minimize this issue. It would be very helpful if anyone can come up with any suggestions (something which I am not already doing) to eliminate the risk of air bubble formation. Thank you!
Hey Shruthi,
I used to have the exact same problem with my westerns. You refer to them as bubble but i can assure you they're not. you're mayor problem is inefficient equilibration of your membrane. Personally I use PVDF which requires activating before blotting but what I discovered was I wasn't equilibrating my membranes for long enough before i made my sandwich. Equilibrate your membrane in cold transfer buffer for at least 10 minutes prior to sandwich building and try bathing your gel in buffer two ~1min, this will remove residual salts which can cause a build up of localised heat on the Gel-membrane interphase. You can rule out any blocking issues as the ponceau is carried out pre-blocking and should never been done once blocked ( if you did, you'll just get a block red membrane, presumably). I wash and soak sponges and filter paper in cold transfer buffer and add ice, cold blocks and water to the out side of the transfer tank, to dissipate the heat as much as possible. this works for me and i hope it help you.
Shruthi,
I am not sure those are air bubbles. They have a very interesting distribution along the membrane as they are perfectly aligned horizontally and vertically. Have you seen this pattern before? In my experience, if a rogue air bubble gets in it looks very circular and is often randomly located on the blot (and never really occurs in the same location twice). I wonder if there is something wrong with your transfer apparatus.
Have you coomassie stained your gel before transfers to make sure the gel looks normal prior to transfer? This would be just a nice control to make sure we know exactly where problem is starting.
~Adam
Hi,
Try using a cold transfer solution the next time, I mean 4 ºC cold solution. Good Luck!
did you look at your nitrocellulose membrane, it might have a problem that affected the transfer and blotting of the proteins at those particular locations. Also, I wonder whay you need such long time for transfer, I've never used more than 3 hours transfer and it worked nicely even with low amounts of proteins.
Another important point to monitor is handling the gel and the membrane, you should not touch them in area of protein blotting. handling is very important to get clean protein blots.
good luck,
As per my experience says, the blot transfer depends on the high tech instrument that u are using like some primitive semi-dry apparatus ,one take 45 minutes for 25mA current and as if u provide the 2 much time then it may lead to further transfer to cotton pad if u use and very little will remain on nitrocellulose membrane. latest instrument like of Thermo scientific as of semi-dry transfer that takes around 5 minutes only. and only use chilled 1X transfer buffer (in methanol) and transfer buffer's activated nitrocellulose membrane for blot transfer. Not much but sufficient transfer buffer should be use and then can use falcon tube as roller (on top of uppermost cotton pad) to remove air bubble. I am sure u will get good results.
thanx
I am used to remove the possible air bubles with a glass rod. It works very well. However I agree with the other colleagues....They do not seems to be air bubles. Check the solutions. If necessary prepare all of them by yourself . Take care to not touch both the membrane and gel. During the transference you should do that in a cold system. Thermal ice pack (Blue cooling unit) inside the buffer tank. And also some ice around the buffer tank. I agree that the transfer period you are using is too long. Usually this can depend on the weight of the protein. But usually maximum 4h are enough. If you are not getting the results, maybe it is not the transference phase, but somehow can be the quantity of protein or the running phase. So check the voltage, time and be sure if your running is going perfect. When you are transfering do a llitle mark in the mebrane pinpointing where it was supposed your protein be marked, I mean the weight of the protein. I do it preferentially on the side that are my controls. It´s easier to not loose yourself when you get the pictures and results. Good Luck!´m sure you will get the results that you wish.
Try transfer with only 2-3 hrs at max rather than keeping it overnight.
Hi I agree with other colleagues, they are not bubbles.
If your nitrocellulose is good another problem could be that if the transfer is hot, the gel itself attached to the nitrocellulose and degraded the protein. This happened to me with a 6% gel. You said that you transfer at 300mA, but, fo me 300mA is very high for an ON transfer. Did you notice if the transfer was hot the morning after? If the transfer is hot you can notice that the gel appear wrinkled, uneven.
Hello all,
Thank you for the responses. Here are the coomassie stained gel after the transfer and the image of the membrane after the antibody incubation. I used a cold transfer buffer. I am trying to study the Poly(ADP-ribosy)lation by monoclonal PARP 10H antibody, and hence the molecular weight of these pADPR chains range from 100-200KDa, and I hence run an overnight wet transfer at the specified voltage conditions. In the past, I've tried running the transfer at lower voltages and lesser time periods,but after numerous western blots, I've optimized the conditions for the western blotting of these proteins as 20-25V, 300mA-500mA, overnight. As, I am using a nitrocellulose membrane for the blotting, I don't think I can pre-treat them with methanol. I use forceps and never touch the membrane with my hand. I also roll out any air bubbles with a teflon roller. I place ice packs on either side of the sandwich inside the transfer chamber and I was concerned that may be these packs are not letting sufficient buffer to circulate around the sandwich. Hence, I repeated the experiment again without any gel cooler packs. Additionally, I laid out individual filter papers, rolled the teflon roller over them to be sure that I am not trapping any air bubbles between the filter papers. When I turned off the transfer the morning after, the transfer solution wasn't hot at all. But, while dissembling the cassette, I noticed that the gel actually kind of stretched a little bit and became very delicate (a sign of drying), which makes me wonder that my sandwich might have been too tight. So, I am repeating it again with 1 less filter paper today. I will post the results after the ponceau staining. Could it be possible that my gel was stretching due to the really tight sandwich and then shrinking back to original size mid transfer? I am attaching a ponceau stained image of the 2nd experiment. This is so frustrating!
can you please upload your previous ponceau images of the same protein apart from already posted here ? and please dont get frustrated.
@anil Raj narooka, umm, other images? It's been a while that i started studying these proteins but I started the ponceau staining my membranes like around the end of February. But it's been a similar blotchy appearance of the membrane, except that's it's not consistently in the same spots. Lol, trying not to. :)
three things you can try : use a thicker gel, and while making sandwich make sure that everything is tighly in contact but not too much compression of gel. Second thing is, while blocking the reaction, make sure you use properly homogenised blocking solution. Also, make sure you use everything as fresh as possible. Third, while making sandwich make sure everything is pre-wet.
Hi there,
These white spots appeared in your membrane are more likely because you prolonged the transfer "blotting " time. I would suggest 3 hours at 110 V ( I suppose you increased the transfer time because your protein of interest is large?). I have also question, Why would you stir during transfer time?.
I always use filter paper to make "the sandwich" . Briefly, I dip the filter paper together with the membrane in the transfer buffer, then place 8 of filter papers in the base---membrane+ gel in the middle and 8 top-pre-wet filter paper (I place these filter paper one by one with ironing between each one) in this manner you make sure there is no air bubble interfere with you transfer step.
Good Luck
Hi, you have all my understanding! The best frustations in my life were caused my western experiments ;p
After I saw the second image, I noticed an improvement, I know that you didn't eliminate all this strange spots (it seemed as something “nibble at” your membrane) but there was an improvement, also in the transfer of high weight proteins respect the first gel (did you do coomassie stained on the second gel?). From your last feedback I can only said that it is really important to wet the membrane, sponges and 3MM paper in the cool transfer buffer, (I also immerge the gel for few minute in the transfer), but you don't have to pre-treat only in methanol. I am concern about the stretched gel after the transfer, did you notice if the gel remain very attached to the membrane, did you have to be careful when you separate gel and membrane, as the gel it too much “glued”? I saw that after you reduced the sandwich thickness you have less “spots” (I use only two pieces of 3MM), I think it is a good point, and I agree with your choice of only an ice pack.
You said that you optimize the ON transfer but if you have this problem maybe it better to make some text with transfer of 1-2h only to understand what is the problem, old nitrocellose, dirty sponges of the transfer equipment, reagent or what else. You have a problem during your transfer reaction but you don’t have a transfer problem, you don’t have to optimize anything else! You have to understand why and what “eat up” randomly your proteins!
PS.You said that you use bolt & mahoney transfer, I read this interesting paper, but I never used the transfer buffer 3 with my SDS or "Laemmli" gels
Hi!
I have trouble identifying your unlabelled images, but its clear that your gel is generally ok. So the problem is the transfer from gel to membrane. Your original concern of bubbles has been well commented to be not the problem. Overnight transfers are common and need not be the cause. You have already taken care not to touch the gel/membrane. At this point I would narrow down to what some others have suggested- try a new batch of membranes that has worked well for others, and make fresh transfer buffer.
April 7th Update:
Hello everyone! I finally figured out how to minimize the air bubbles during western blot transfer. It was something about my skill regarding the western blot sandwich set up that I had to work on. Apparently, while closing the sandwich cassette's clamps, I was holding the cassette in such a way that a gap (which was very difficult to notice) was being induced between the membrane and the gel, and any air bubbles in the vicinity were creeping up into this gap. But, yesterday, based on the suggestion of my labmate, I held on to the sandwich cassette very tightly with my dear-life (trying not to induce any gaps) and voila! No air bubbles (very minimal and not in the region of interest). I should probably work further on this way of handling the cassette to eliminate the issue of air bubbles altogether. Thank you all for so many troubleshooting tips and advices.
Hey Shruthi,
I used to have the exact same problem with my westerns. You refer to them as bubble but i can assure you they're not. you're mayor problem is inefficient equilibration of your membrane. Personally I use PVDF which requires activating before blotting but what I discovered was I wasn't equilibrating my membranes for long enough before i made my sandwich. Equilibrate your membrane in cold transfer buffer for at least 10 minutes prior to sandwich building and try bathing your gel in buffer two ~1min, this will remove residual salts which can cause a build up of localised heat on the Gel-membrane interphase. You can rule out any blocking issues as the ponceau is carried out pre-blocking and should never been done once blocked ( if you did, you'll just get a block red membrane, presumably). I wash and soak sponges and filter paper in cold transfer buffer and add ice, cold blocks and water to the out side of the transfer tank, to dissipate the heat as much as possible. this works for me and i hope it help you.
Hi Shruthi, I'm having the same problem. I've tried running the transfer at 4degrees and tried letting the membrane soak in cold transfer buffer as well as rinsing/submerging the exposed gel in cold transfer buffer. I think you're right in that it has something to do with the way you clamp the sandwich. Can you elaborate a bit further on this. Is it a matter of force? Or do you need to apply even pressure and keep that pressure until its in the tank? Thanks
@James Firth: I zeroed the reason in my case as: the heating of the gel due to prolonged transfer time at high voltage run caused the gel to attach to the membrane and when the sandwich was dissembled, this fusion removef the transfered proteins haphazardly. I tried reducing the transfer time and I didn't see significant splotches after that. Good luck!
Interesting, I just ran my entire 1hour transfer at 20V in a 4degrees cold room. Maybe I'll try cooling down the gel for 10mins before constructing the sandwich. How long do your transfers last. Thanks again
I also faced this kind of problem and later fixed this issue by replacing the sponge.
Hi, I was having this same problem and realized the sponges were not good. I temporarily solved this problem by allowing the sponges to soak in cold transfer buffer for ~30 min before setting up the sandwich. The sponges should be replaced.
I used to do only dry transfers but when I changed to wet transfer (methanol/no SDS) I had an identical problem - changing the sponges did not work for me. what ended up working was doing everything to make sure the apparatus was cold throughout the transfer. as mentioned by folks above the problem is not caused by air bubbles but instead heating of the gel. To keep things especially cold, you can soak the sponges ahead of time, transfer at a slightly lower voltage (80V instead of 100V) for a longer period of time, and perform at 4C. This should ensure that the transfer buffer and apparatus remains cold. Ice block and stir bar are also critical to make sure there is no localized heating of the apparatus on the side opposite the ice block.
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