Dear Mohamed

There are a number of things that could be going on here! Have you stained any gels with coomassie blue to check that the problem is during the transfer and not in the electrophoresis?

1 I would suggest that you make sure that your wells are washed out thoroughly with running buffer, as bits of detritus will affect the running of the gel.

2 as others have suggested, you could use a prestained marker, it also helps to see how you gel is running.

3 I would also make sure that you run your gel slowly until the BPB dye has left the stacking gel, since this stage is where the isotachophoresis occurs and you proteins begin to separate, and makes the final bands sharper, and then run the second stage at a lower voltage.

4 When you do the transfer make sure that the buffer does not get too hot-if it does, just change it (if it is a wet transfer)

5 After the transfer try staining your membrane with Ponceau Red which will tell you if the transfer is effective, and if your bands are smeared/wobbly. You can also stain the gel with coomassie blue-the very high molecular weight bands should not be smeared or wobbley if it is the transfer that is at fault.

6 I noticed that the autorads are a bit dirty - If you are blocking with BSA this can often happen -I make up a batch in TBST with sodium azide in, and then filter it using a 0.2 micron tissue culture filter as this cleans up the blots, and the BSA solution can be stored in the fridge for ages.

7 One thing that really affects transfer is non ionic detergents, the most common source is washing up liquid or tween. I leave my whole tank with sponges in, in the sink with cold water running into it for at least 10 minutes before I equilibrate the sponges in transfer buffer to clean it.

I hope this helps

Dear Antar,

your problem was quite interesting. I am surprised not to see any signals in middle. Actually, this happened with me in past. I suggest you to run two gels in parallel and transfer one of it to membrane and the other one for CBBR staining. Then you will come to know whether the problem in running or western.

As you are sure about the reagents, protocol, I suspect that there will be a problem with your samples. In real, the protein extracts you made may contain high salt or lipids or some other reagents used in your extraction may interfere with protein running and to western signal.

If you check all other things carefully, you may find a solution in soon. Otherwise I will suggest you to ask your other lab mates to do same thing for you once to get rid of this problem.

Good Luck.

Very odd...Since the buffers seemed to have worked with others I can only suggest to double check the voltage on the transferring machine and the gel electrophoresis apparatus with others in your lab. Other than that, I would say take another look at your gel preparation and efficiency in protein loading (although this may be unlikely you never know)

Did you permeablise the membrane with methanol before transferring?

no, should i do that Jake Shortt

Dear Antar, L

As Upendarrao Golla says, I suggest you to begin by a coloration of your gel before blotting procedure to be sure that the problem is not your gel or your samples. Secondly, is your membrane same as your colleagues in the lab (PVDF or nitrocellulose??)?

Why don't you ask a colleague to run your samples on one of their gels and do the transfer and see if it fixes the problem. If it doesn't you know its a problem with the preparation of the lysates etc. Otherwise you'll have to watch carefully to see what you're missing.

Didiers point is a good one - check you are using the right membrane. I usually permeablise for 30s with 100% MeOH. What is the methanol conc. in your transfer buffer?

Yes i use nitrocellulose

No, nitrocellulose membranes don't need to be pre-incubated with methanol. PVDF membranes have to.

Is it posible to get one of your colleagues to have him watch you while you blot? Sometimes another person spots mistakes easier thank you do yourself.

How do you blot, semi-dry or in a tank? Is it possible that you mistakenly reversed the polarity of your powersource (the gel should always be at the negative elektrode)?

Dear Antar

I already had the same problem, but in my case I discovered that the problem was a poor contact in the power of transmission. We did buy a new equipment and then the problem was solved.

I agree with above mentioned suggetion and I think you need to stain your gel before you go for transfer. If this is worked and running the gel has no problem then you can stain membrane with Ponceau to see if transfer was done.good luck!

Did you check the membrane with Ponaceau S staining after transfer???

In my experience, smears appear when the quality of gel has degraded. If your using precasted gels, this will happen especially if you use gels well past the expiration date. Additionally, smears can also come from running the gel at a high voltage (the gel become warmed and may warp, distorting the bands). Make sure to flush out the wells before loading the sample as well, especially if the buffer used to make the gel is different than that of the running buffer, the difference in salt content, pH, etc. can change the migration of the protein down the gel. Hope this helps!

Yeah, it looks like the problem maybe happening before transferring, i.e. your gel is not running properly.

Make sure your bands are nice and sharp by staining a gel without transferring.

I am not sure how you prepare the sample.

One of the lab techs in my old lab had this problem of protein smearing. The protocol in our lab said that you need to have a PBS NO BSA wash before the lysing step. And I notice that if you forget this step, the proteins tend to smear. The BSA in the wash buffer tends to alter the run of the samples.

try running a prestained marker and see how it transfers. If the prestained marker transfers well but not your samples, it may be your sample preparation. If the prestained marker does not transfer well, check your protocols and try making new buffers.

BTW: are you reusing your transfer buffers ?

I do not completely know what is causing your bands to smear, and without the details of your protocol I will offer you some tips:

1. The high background is likely due to excess developer. I literally take paper towel and dry the excess ECL off of the membrane, and the background is always clear.

2. I don't know your size of your protein, but wet-transfer works better for larger proteins. Also, I find its better to use cold transfer buffer no matter which method you use.

3. If you make your own gels, make sure to mix them well (meaning vortex!). Yes there will be bubbles, but you need the APS and TEMED to be distributed evenly in the sample. It certainly helps make my gels very pretty.

4. What voltage do you run the gel at? We maintain ours at 25 mAmps (for 1 biorad minigel) to not overheat and fry the gel.

Best of luck!

If you followed all of the above and still your bands are smearing, try cooling during the gel run.

Some brand of geles cause the bands to smear. Other possibility is the temperature during the running. The running buffer should be cold to avoid high temperatures during the running. Don't run at high voltage, that also cause the bands to smear. I would like to know if you buy the gels or make them yourself. If you make them, is better to take all the air out of the mix acyl/bisacr with vacuum for 3-4 min.

If you gave more details about your protocol, it would be possible to find out what the problem is. Good luck

Are you all using the same sponges to build the transfer stack? If you by chance use dirty sponges, this could result in smearing during the transfer.

We also noticed in my institution that SDS grade could also influence run badly. Check you did use electrophoresis quality one

I would recommend that you pay attention to the BSA Buffer (may be use fresh) and try reducing the SDS a little. However, I need to know what the protein sample was for Western blotting.

Dear Mohamed

There are a number of things that could be going on here! Have you stained any gels with coomassie blue to check that the problem is during the transfer and not in the electrophoresis?

1 I would suggest that you make sure that your wells are washed out thoroughly with running buffer, as bits of detritus will affect the running of the gel.

2 as others have suggested, you could use a prestained marker, it also helps to see how you gel is running.

3 I would also make sure that you run your gel slowly until the BPB dye has left the stacking gel, since this stage is where the isotachophoresis occurs and you proteins begin to separate, and makes the final bands sharper, and then run the second stage at a lower voltage.

4 When you do the transfer make sure that the buffer does not get too hot-if it does, just change it (if it is a wet transfer)

5 After the transfer try staining your membrane with Ponceau Red which will tell you if the transfer is effective, and if your bands are smeared/wobbly. You can also stain the gel with coomassie blue-the very high molecular weight bands should not be smeared or wobbley if it is the transfer that is at fault.

6 I noticed that the autorads are a bit dirty - If you are blocking with BSA this can often happen -I make up a batch in TBST with sodium azide in, and then filter it using a 0.2 micron tissue culture filter as this cleans up the blots, and the BSA solution can be stored in the fridge for ages.

7 One thing that really affects transfer is non ionic detergents, the most common source is washing up liquid or tween. I leave my whole tank with sponges in, in the sink with cold water running into it for at least 10 minutes before I equilibrate the sponges in transfer buffer to clean it.

I hope this helps

When running the gels and performing electro-transfers in a 4C slide fridge - be certain the whole unit is sitting atop a magnetic stirrer - with a stir bar at the bottom of the tank during both gel run and electro-transfer. Stirring a magnetic stir bar in the bottom of the tank (@ ~360 rpm) helps distribute heat and keep the running and/or transfer buffers homogeneous - be sure to use -20C pre-cooled ice pack as well during transfer if you have one.

Not sure if working at room temp helps - but, perhaps in some situations it does. Transfer to membrane generates more heat - so definitely run in the cold for that. Overloading sample is another possibility for smearing.

But - as Andrew Sunters said above in his point #1 -- be sure to pre-wash the wells out gently (e.g. with a 1 mL pipet) with running buffer so gel storage buffer and/or any un-polymerized gel material is cleared out.

Another major Achilles' heel is forgetting to soak the gels in transfer buffer (after gel run) for at least 20 minutes minimum (to rid entirely of SDS) prior to transfer. 2 or 3 minutes of soaking gels in transfer buffer is not enough time to ensure the complete removal of SDS from the gel.

Also, pre-running the gel for 5 to 7 minutes at 100V after cleaning the wells with running buffer (before loading samples initially) apparently makes all the wells better 'starting gates' too; keep all your horses happy and give them all a fair shot at the gate.

Most of the time what goes wrong is in the sample preparation. Also as rightly pointed out by Andrew, you need to see your gel closely and make sure, it is in good condition. I would further add to check your power supply too...

Your marker and your protein bands being smeared suggest that something is wrong in your running or you transfer. I therefore suggest checking every steps if you are using the same protocols as your colleagues and yours is not working (it means you need to identify the steps that you are overlooking. (Western blot are easy enough to be fail if you are not careful).

1-Make sure you do not load too much proteins. Calculate carefully your total protein content.

2-Double check and make sure the gel % and thickness is appropriate for the voltage and running time you are using.

3-Prepare all you buffers fresh and mix them thoroughly before pouring them.

4-Make sure your gel is not overheating during running or transfer. Cool down your transfer buffer before use, use ice pad, place the transfer apparatus in an ice bucket.

5-Rinse very well with distilled water all your blotting equipment before use ( especially foam pads to remove any trace of detergents from previous runs)

I am curious to know if after one month the problem is solved now :) and what was the problem!

I see a lot of good ideas of what´s going on.

First I suggest to run the electroforesis in a low rate. 90 mV will be great. Its a little bite longer the time of the run but it would be better the run 4 or 5 times more.

Then I suggest that before you transfer your gels to the PDVF or Nitrocellulose membrane you put the gel one or two minutes in the transfer buffer. This would clean a little bit of the dirty of the crystals of the electroforesis. Even when it seems that they are clean they may not.

Another tip, if you want to call like that ; is to put both abs with TBS- tween or with PBS-tween.

The last one I have is that if you see that it have to much signal when you are revealing; you could put a litlle bit of TBS-tween or PBS-tween with a little hand shake for two or three seconds and than reveal it again. You would see that the siganla is a little bit less and you could work better.

I hope my tips are helpful,

Good luck.

Pinto-Almazán Rodolfo

Dear Mohamed,

I was just looking at your question again, and I might suggest on reflection that the bands are not really smeary per se, but are wavy or rippled. In my experience I would suggest that if you are doing a wet transfer that your buffer is getting too hot- this sometimes leads to a ruffling of the gel onto the membrane sandwich with heat, and this leads to the rippled effect on the bands as they transfer. I would suggest, as others have, that you do not transfer at such a high current, and maybe even used chilled buffer of an ice pack in the transfer tank. The high background in the films is also dealt with here and I suspect is more to do with the blocking/overlay etc. rather than the transfer.

Hope this helps

It is interesting that this happen to my colleague the past 3 days. She ran the electrophoresis twice using BioRad 10X transfer buffer but lost all the bands. She used Ponseau stain and could not see anything on the membrane. She ran the 3rd time, using excatly the same method, and asked me to be by her side during the transfer then it worked. I do not believe she changed anything since she is an experienced researcher. My hunch is there was a mysterious fluke. However, do make sure you have enough methanol in the transfer buffer and make sure to use a roller to get rid of air bubble between gel and membrane. Thirdly, make sure the sponge is able to hold up the transfer sandwich in wet-transfer.

I read all the comments, almost every thing that might affect the transfer is mentioned. I am not sure if is due to Biorad transfer tank. In my lab we recently bought Biorad tank and the problem start, then we found out other labs using this device have same problem. It looks that instead of running at constant voltage (100) is better to run at 30-40 V for 3 hours to get good transfer from gel to membrane.

i started to have the same problem recently, when i run a 7% gel my ladder became smeary. I suspect if it is the gel or the running buffer?

Recently, i found that bands were smeared because of gel problem (APS should be freshly prepared).

it was APS problem Jia Wei, check whether your APS is freshly prepared or not

maybe...the only time i found this problem is that I used 10% APS one day old

yes, Jia Wei

Could it be an electrophoresis problem? If you Coomassie-stain the gel rather than transferring, do you also see the waviness in the bands? The Coomassie stain should help you isolate the point in your protocol where this is happening. Does your protein have post-translational modifications? I've seen smeary bands caused by glycosylation.

I would rather suspect the buffer quality for the problem. APS buffer would be my major suspect. Replace it with some other brand. May be it will help .

Everyone, I have a problem in my band. It occurs at high MW about 125 kDA in this picture while a band in low MW (not have a smear band). The protein is a phospho form.

I lyse protein in NP-40 buffer with protease and phosphatase inhibitor and then centrifuge at 12000 rpm at 4 C. I run 10%gel at 90 v about 1.30 h and tranfers on ice with 100v about 2 h. Blocking with 5%BSA in TBST and 1 AB 1:3000

Whether B-actin is ok.

I saw that smear band look like V-shape.

Thanks you so much

Suwadee Phowichit

Hi Suwadee,

I took a look at your picturre and increased the light intensity. For me it seems that you also the bands with lower MW show at light smear. Can you tell me how many Protein you load on the Gel, maybe it is to much material, or the gel gets to hot during the run. I run a Proteingel with max 25-30 mA at RT, with higher mA I would put in a cold room. And for the tranfer I take 3mA/square centimeter gel szize, at 4°C for o/n.

Dear Sibylle Szabowski

I used sample protein just 20 ug/lane. Recently , I solved the problem from Cell Signaling Technician support by sonicating protein about 15 s * 3 times but my band still happen a smear problem , while in low MW about 43 KDa doesn't has a problem.

My western blot conditions were keeping the sample in NP-40 lysis buffer and sonicated with 15 s *3 times, and then protein sample centrifuged in 4C 12,000 RPM about 15 min.

,

I used 10%gel polyacrylamide gel, ran in 70 volt 30 min and 100 volt about 1 h, Wet transfer with 100 volt about 2 h in ice cold condition, and used 1 Ab pFAK with 1:3000 , 2Ab 1:3000. All of the buffers do not reuse and freshly prepare. The B-actin band is ok.

Thank you so much for your suggestion.

Hello everyone. I have recently been having the same problem, just a little different. My target protein is about 25kDa and I have been successfully using a 10% prestacked Bio-Rad gel. All buffers seem to have been working OK until the last month. Now, with all the same conditions, ONLY THE STANDARD MARKERS get transferred onto the membrane and I dont see any of my samples at all. Last week I did a Coomassie stain of the gel following transfer and I found that the lane with the marker had vanished while the rest of the lanes were nicely filled with the samples (see picture). The two lanes with the arrows are where the markers WERE. They are the only ones that appear in the blot below.

Some troubleshooting guides say that there could be some precipitation of proteins and therefore suggest adding SDS to the transfer buffer but my transfer buffers already have SDS.

My primary antibody dilution was optimized and has always been at a dilution of 1:200 (goat) and I use an anti-goat IgG secondary at 1:2000. It seems to me that there is something wrong at the detection level. I tried using a different primary that I had used with the same samples before (successfully) but this time that doesnt appear either.

Would appreciate any thoughts or advice.

Thanks

Hi Donald,

have you tried just an antibody that you normaly took for loading control?

Due to my experiences a transfer is never 100% so you will always have some protein left wich is stained by Coomassie.

If everything worked well until last month, then I sugest that there was a change in something.

In our lab we found that changes in the quality and the pH of the water can make a lot of trouble (eg new or to old ffilter to demineralize tap water).

Best wishes

Sibylle