From the gel end - it is possible that the sample ran poorly through the gel. Was the gel pre-cast and near expiration? Was the gel cast yourself? Using fresh APS? Too little TEMED?

From the transfer end - What type of membrane? Uneven blots such as these can be because a PVDF membrane was not completely pre-wet with methanol. Could be insufficient volume of primary or secondary staining solutions, air bubbles, or poor reagent quality.

The gel was self cast and made fresh, looking good. The PVDF was properly pre-wet with MetOH. I believe this is not uneven transfer - I believe there is more protein in the bands on the left. What is strange is the particular band shape...this is not a common smiling... Beta actin for this is looking quite good... Is it because there is not enough protein? The blot was incubated with the film for 2 minutes so not that very long....?

If you exposed this blot for only 2 minutes then it may be too much protein rather then too little. I would suggest looking at the gel before transfer to see how it ran - some of the flourescent protein stains will allow you to stain protein without first fixing the get and then can be destained - then you can run the transfer.

Maybe there are some bubbles between your gel and membrane during transfer step .

As the Beta actin bands looks good, your protein transfer should be OK. You may have to look at the volume and addition of ECL reagent to your blot before you record the luminescence on a X-ray film. I have similar experience when I put very little ECL reagent (~2mL) over the blot placed over a saranwrap sheet ( the reagent spreads unevenly over the blot). So you get good signal at some places and improper bands. In such a case, you may need to incubate the blot in at least 6ml of ECL reagent in a glass tray such that all the places over the blot get uniform distribution of ECL reagent.

Hi,

there are a few reasons for this:

1) large volume of sample was loaded - use less volume and concentrate sample if too dilute

2) too little volume is used causing uneven distribution on surface

3) sample not loaded symmetrically, that is, evenly - ensure proper loading

4) the surface of the resolving gel is uneven - can be a problem with home made gels, pay more attention when pouring

5) the gel is old - make fresh gel

6) migration is too fast - slow down run

7) migration too hot - run on ice

8) not all wells are loaded with the same volume - load all wells with same volume. Also, if running an empty well, add equivalent amount of running buffer only.

To me it looks that different volume of sample was loaded because of the "blobs" of protein on the right side of the picture.

Hope it helps

Hi

stain the gel after the transfer so that u will get to know weather the transfer was complete or not.

gels should be prepared proper so that u will get the proper banding.(Avoid uneven surface)

while keeping it for transfor avoid air bubble.

when u r adding final substrate make sure that the substrate spread properly on the membrane.

load equal volume of the sample.

good luck

I agreed with some points of last answer:

4) the surface of the resolving gel is uneven.

Clean very well the glass plates before use, add always fresh APS, in this case you have to wait 30min for running gel polimerization and 15 min fot the stacking one. I made often the gel the day before

6) migration is too fast - slow down run.

I absolutely agreed with this point, I have a lot of running problems with posphorylated protein, orrible bands shape appear in my gels because I did not run at an appropriate voltage. There are a lot of interesting discussions to this point in reasearchgat;e, smile band are often caused by running too fast.

7) migration too hot - run on ice.

I did not have this problem during the run, if you find pour running buffer became hot check the buffer recipe or the pH

8) not all wells are loaded with the same volume - load all wells with same volume. Also, if running an empty well, add equivalent amount of running buffer only.

Very good advice! For the band at the righ side of the gel I want to ask if you check the band after transfer, with Ponceau, if were good and loaded same amount, the problem can be uneven covering with antibody or ECL as pointing out in the other answers, if not the problem ca be the loading.

I've seen this on one or two of my membranes - dumbell-shaped bands are not caused by a transfer or detection problem.

As Valeria suggested, slow the run voltage down and/or run the gel on ice.

If the problem persists, consider adding antioxidants to the cathode buffer or using a different running buffer all together.

Best of luck!

Thank you for the suggestions but this is not a problem with typical smile its rather dumbell shape mentioned by some of you. Ponceau staning of PVDF after transfer looks fine. Today I checked it once again - used higher concentration of secondary antibody, developed it with different ECL substrate. The effect is that with Pierce company it is slightly better than previously used Advansta ECL but still the bands are irregular. Could it be a problem with old secondary antibody? Today with 1/3 higher concentration of secondary I see more background and slightly more regular bands but they are still unsatisfactory to estimate the differences with no doubts. The antibody is two years old but it was aliquoted and store in small volumes at -30.

You suggested running SDS PAGE slower but - the buffer was not hot after electrophoresis? Is it possible that being at room temperature it is still to hot for proper electrophoresis ? (It was 10% gel)...

Though it may not "feel hot", temperature changes or oxidation in running buffers can alter the charge distribution of the buffer counter-ion. That effects the electrical field (you're working with a circuit) and how your negatively charged SDS micelles migrate. The barbell shape could be due to temperature-dependent differences in sample mobility or the running buffer itself.

Personal preference, but started running everything in chambers packed into ice. I have not seen barbells since. Might be worth a shot, or use different buffer.

Hi Tomasz i am sorry that you can not resolve your problems yet!

For the running problems (for the left side of the gel) Grant gives use very good advice, and for sure you can improve your results. If you can ask help to your coworkers use other buffers to make some test. Check also if the apparatus was clean of acrylamide residues that can caused irregular running (sometimes you have to be a little paranoid!).

The right side of your PVDF was the one that make one to think that something strange happen. I mean your background are really clean if the problems was the antibody why you have this results only on the right side? If you have doubts about the antibody (you mention only the secondary I suppose the primary is ok), try a new one, if you can, as you try a new ECL, but I think that first of all you have to revolve the dumbell shape and then the blotchy problems. Maybe the first problem can resolve the second one.

As other mention before pay attention to the volume of antibody incubation and to the velocity of your agitator.

Good luck

PS: I am sorry because I was not accurate I mean that smile and even dumbell shape bands can be caused by high voltage.

I asked you about staining Ponceau out of curiosity, because I also have problems witn irregular band shape and my Ponceau was "goddam" perfect!!!

Thank you for advice I will start running my gels in the coldroom if this could really help me to achieve more regular bands. I think I just need to do this blot once again. The buffer was quite old but the other blot from the same electrophoresis in that chamber was ok so I still do not know what was wrong - probably it was just a bad lunar phase:)