Dear Abdul,

I also experienced a similar problem once. The gels had run so beautifully, so I was surprised to find that the proteins transferred in a wave-like pattern onto the membrane. I tested a number of possibilities, but in the end it was the thickness of the filter paper (we had purchased a new batch which wasn't quite the same as we had before). All it took was to add another thickness of filter paper or use sponges that were thicker. And viola! The mystery was solved.

Hope this helps!

Maria

Did you presoak the PVDF in methnol for more than 5 minutes before putting the gel on to transfer? Make sure you did not mistakenly use running buffer for transfer which is not uncomon at least to some of my students.

Abdul!

in most of the cases, such blurring during transfer occurs when we do not wet the PVDF membrane in pure methanol for 30 sec to 1 min followed by keeping it in transfer buffer for 5 min. After this use the membrane for transfer in pre-cooled transfer buffer.

Abdul,

In advice to earlier contributors advice I would like to mention few more things.

1. Check the MeOH % and grade. I always use 10% (V/V), 20% seems to be high.

2. Check the current or voltage. Preferably 100V for 1h with cold pack and constant stirring by a magnetic bar. If possible run the transfer at cold room or cover with ice.

Hope that helps.

One quick thing to check if proteins were properly transferred onto the PVDF the membrane is to stain the membrane in the Comassie Brilliant Blue staining reagent, which is maybe the most widely used visulization methods for proteins in the SDS-PAGE, and also works well for the PVDF membrane. Just stain it for 3-5 min, and then wash the membrane with the destaining buffer.

Dear Abdul,

May be because of the second step, you faced the problem. After making the sandwich, one should not wait. Did you run the transfer at 4 degrees or in the cold room or with the ice pack in it? Generally running the transfer for 2 hours at 4 degrees at 100 Volts is recommended. Otherwise, you can also change the membrane. You may use nitrocellulose membrane instead of pvdf because the main problem in pvdf membranes is activation. Sometimes charging of the membrane with methanol goes wrong and in nitrocellulose, there is no need of preactivation and you can check the transfer by staining the membrane with Ponceau reagent.

I hope this helps.

Best Regards,

Dear Abdul,

I also experienced a similar problem once. The gels had run so beautifully, so I was surprised to find that the proteins transferred in a wave-like pattern onto the membrane. I tested a number of possibilities, but in the end it was the thickness of the filter paper (we had purchased a new batch which wasn't quite the same as we had before). All it took was to add another thickness of filter paper or use sponges that were thicker. And viola! The mystery was solved.

Hope this helps!

Maria

Dear Abdul,

I had something happen like this with nitrocellulose, but perhaps it could also happen with PVDF. I made the gel according to a recipe I had always used in a previous lab. The new lab used a different SDS-PAGE recipe, and they did not use standard western transfer buffer either. There was an incompatibility issue between how my gel was made and this transfer buffer. This created way more heat than 1 hour at 100V should produce and was melting the gel and overheating the nitrocellulose.

Have you used this gel recipe and this transfer buffer before?

Stacy

Thanks all of you for sparing sometime to answer my query.

@ Dear Stacy Thanks for your answer,

Honestly, this was the very first time that I am doing WB and I use the standard 12 % 1.5 MM thick SDS-PAGE recipe mentioned on gel calculator (http://www.cytographica.com/lab/acryl2.html) link. While all the buffers were made as mentioned by Abcam WB method.

Even though I take off cassette after an hour of the run was completed and whole running assembly was surrounded by ice and an ice pack in it, the transfer buffer was luke warm.

It is interesting to learn so many different experience. A few things I would like to add

1. The methanol can be 10 or 20% and I have tried both and they all worked. Use 10% maybe more environmentally friendly. Or you can transfer without methanol but fixed the membrane afterwards with ethanol or methanol.

2. Once the gel is in methanol containing transfer buffer the proteins are pretty much fixed in the gel so some delay in starting the electrotransfer should not be a big problem. Also, the current will be off after the preset time for most power pack and leaving the membrane in the box for 80 minutes should not be a problem either.

3. Thick filter paper or another pad of sponge sometimes may not help since the center part will be buldging outwards and make the transfer of that portion less efficient.

I have used both nitrocellulose and pvdf membranes for western transfer. i prefer pvdf as they last longer than the nc membrane. i think presoaking for 30 sec to a min in methanol is sufficient activate the membrane. As told by previous contributors leaving the sandwich in transfer buffer shouldn't cause much problem. One of the problems could be lower SDS content in the running buffer of the gel. I once faced a similar problem with the electrode buffer lacking SDS and the bands were streaky. Also to visualize proteins on membrane i suggest ponceau staining after transfer instead of comassie. Ponceau is a reversible stain (and can be reused many times) you can wash off the stain from the membrane using TBST (10 min).

Hope this helps.

1.5mm is a very thick gel (0.75-1mm is more regular). Is there a reason it needs to be so thick? Is your sample dilute and you need larger wells to load enough? I ask because a gel can hold vastly more protein that you can blot on a membrane, but this is not likely your problem. As many have already said, it was the waiting time that is the likely problem. The well focused protein bands will diffuse over time in the gel and this may explain what you are seeing. Also, wet transfers are more likely to have random problems (in my experience) whereas semi-dry transfers are more reliable and can be performed in 10-15 mins with the added bonus of being able to transfer to two membranes on either side of the gel for a mirror image of the protein on each membrane.

Thanks Matthew Padula for your suggestion!

I understand 1.5mm is quite thick, but my cell lysate has 1.5 ug/ul proteins conc and I want to optimize protein of interest at different conc. So I need larger volume to run.

I guess you are right the problem was due to over-waiting, as I transfer proteins today once again without any change in my previous protocol. So far it seems OK and I got nice protein ladder band on PVDF membrane..

Hi Abdul,

It sounds as if the device was overheated during the transfer. Overheating can cause dissortion in the bands and give you ugly looking western blots. Soaking PVDF in methanol for 5mins should not be a problem (make sure that you wash excess methanol by incubating the membrane in transfer buffer after activation, a step that I think is quite important). Not removing the sandwich from the tank after the run should not be a problem, as long as the sandwich is still wet and not left to dry. The issue you had with the device looks more suspicious to me. I do not know the voltage you run the transfer under, but as I said dissorted bands indicate overheating. Try to keep a low voltage when transfering, I do not exceed 80 and always place a block of ice in the tank to prevent temperature from rising. Also, always make sure to equilibrate your gel in the transfer buffer prior to transfering to prevent gel shrinking.

Hope it helps

You did not say what molecular weight proteins you were looking at. For many proteins, you can omit the methanol completely to aid transfer. If using low molecular weight proteins, make sure you are not transferring to long as it can lead to bleed through. Always make sure that you use chilled solutions and run the transfer cold (either in an ice bucket or in a cold room). Once the proteins have transferred, leaving them in the gel apparatus is not a problem.