As Irene Reimche and Pierre Béguin mentioned, air bubbles is a likely candidate. It also looks like there was some movement during the blotting process where the membrane partially slide relative to the gel. Lastly it might be the membrane was not thoroughly wetted before using. Be sure to read the instructions on how to properly wet the membrane as it varies a lot depending upon which membrane type you are using.

Hey Andrea,

I haven't seen something like this before. Did you check the blotting before immunostaining with Ponceau? What about air bubbles during the blotting?

Best regards!

It looks like poor contact (air bubbles ?) between the gel and the membrane. The blurred area at the top could be due to some shifting between the gel and the membrane. I would try running the blot again, making sure to lay the membrane on top of the gel in one stroke and to avoid bubbles by wetting the gel with transfer buffer prior to laying the membrane.

As Irene Reimche and Pierre Béguin mentioned, air bubbles is a likely candidate. It also looks like there was some movement during the blotting process where the membrane partially slide relative to the gel. Lastly it might be the membrane was not thoroughly wetted before using. Be sure to read the instructions on how to properly wet the membrane as it varies a lot depending upon which membrane type you are using.

Did you use commercial gels? Were they not expired? And I guess you used clean reagents without any particles in them? I mean, wouldn't air bubbles more round?

Regarding the lower picture: it could also be that the membrane was compromised. If you use PDVF membrane it needs activation with methanol (100%). perhaps you could check if that step, if you use that membrane, was carried out well enough. When in doubt, prolong the soaking in the methanol.

Bottom gel is air bubbles definitely. You need to make sure to wet the blot completely in methanol and then in transfer buffer. Then when you overlay the blot on the gel I generally use a plastic pipette and roll the pipette over the blot to make sure I get all the bubbles out. The top gel looks like either air bubbles or the blot somehow moved during transfer. Make sure you add enough sponges so it is tight and the blot would not move during the transfer.

I noted these blurs right after the blotting, but before, during the sandwich assembly, I didn't noticed air bubbles between membrane and gel, however I rolled on the paper sheet after putting it on the membrane. Before the blotting PVDF membranes were activated in methanol 100% for 30 seconds/1 min and then left soaked in milli-q water for a while (about an hour). Gels for the protein running were made by us. All these same materials are commonly used also by other colleagues and they didn't noticed such problems

Perhaps the gel ingredients didn't mix well or gel solidification started before you pour the gel? Or could be that the glass sheets you used for preparing the gels had some fat/fingerprints on them.

Hi Andrea Cioccoloni, the top blot must have resulted from a shift during the transfer while the bottom blot must have resulted from bubbles (as widely suggested by others).

Suggestions: Ensure that the cassettes you use to fasten the gel, membrane, filter papers and sponges hold these items firmly before starting your transfer (for wet transfers) or screw your transfer pack firmly (for semi-dry transfers).

As for the bubbles, wet your membranes properly and after setting up the sponge-filter paper-membrane-gel-filter paper-sponge sandwich, use a "roller" to press out the bubbles that might have formed between the gel and the membrane.

I hope you find this helpful.

Hi Andrea!

The top blot seems to be a result of shifting in contact between your gel and membrane. What kind of transfer methods are you using? The transfer sandwich should be properly tight to avoid these kind of incidences. Also, make sure the transfer is carried out at proper voltage and current to prevent overheating.

The second gel seems to have either bubbles Or fragments of gel blocking the transfer of proteins from gel to membrane. After preparing the transfer sandwich, you can use a test tube as a roller to drive out the bubbles. The others have suggested few other important points as well. Hopefully you will be able to troubleshoot this. Good luck!

Andrea Cioccoloni

When put gel and membrane together , things to be noted:

1. How much time do you give the membrane to get fully soacked?

2. Please make sure that all the bubbles should come out before tightening the gel and membrane to ready for trans blotting. Do you apply some gentle pressure.

3. Make sure that the gel and membranes should not be touched from inside while performing this process.

I am sure you will get great results. It seems from the photograph that you have already electophoresed the proteins nicely.

1. Could be inefficient soaking of membrane prior to use as stated previously

2. Membrane has previously been contaminated prior to use

3. Dirty transfer apparatus: clean all equipment and use fresh transfer buffer

4. Dirty membrane post transfer i.e. old gel sticking to membrane

5. Dirty developer/fixer (if using)

6. Air bubbles? but they don't look like any air bubbles I have seen before

7. Dirty hypercassette (if using)

8. Problems with blocker and/or buffer: particulate matter present. We find this with milk protein sometimes. Make sure things are fresh and well mixed