Does anyone have any experience with wet transfer? When I use a the transfer buffer to wet transfer my blot, the marker bands 'smudge' across the membrane. They do not remain sharp, and therfore when I develop my membrane the bands for my protein are all smudged and not clear.
To prepare my blotting buffer I use:
8.7g Glycine
17.4g Tris-base
1.11g SDS
600ml Methanol - which i then make up to 3L using distilled water. I transfer my blot at 100V for 60mins.
Thank you
The pH is important. In the gel you have pH 8.3, in the transfer buffer pH 8.8.
This means, that you have first an isotachphoresis before any protein moves out of the gel. This is a prerequisite for sharp bands.
But ... it's much more important to work fast. End of the SDS-EP, there is no need for gel equilibration, this takes time (and the proteins will use this time for diffusion ... and this will lead to non-sharp bands). Build you package filter paper, (NC)-membrane, gel, filter paper with prepared material. Everything (except the gel) should be wet, free of air bubbles. It's should be done in less then 2 - 5 minutes.
... and due to the isothachophoresis, let it transfer over night at 15 °C. SDS in you gel can crystallize at lower temperatures ... and your band will be not sharp again.
The wet transfer should be performed in a cold chamber, it generates much more heat than semi-dry, your problem will be solved
Dear Ayah Siddiqi, could you post a picture of one of your blots. Are you shure your gel still has sharp bands? How large is your protein of interest? From your buffer and your transfer conditions I cannot see anything wrong. Best wishes, Erik
I use:
5,8g Tris
2,9g glycine
4g SDS
up to 800ml
and 200ml of methanol
important is a temperature(run with ice in cold room) ! It is also a question how big is your protein, I have small protein and I run transfer for 90min and 350mA.
good luck !
I don't use SDS in my transfer buffer- I also usually transfer at 35V for 2 hours with cold buffer. The lower voltage and temp seem to help with the smearing. good luck!
Hi Ayah, what is the mol. weight of the protein of your interest? It will be useful to see the blot for accurate suggestions. Anyway try to avoid SDS in the transfer buffer, as it might cause some problems to proteins while transferring to the membrane. Literally the SDS from the gel will go in to transfer buffer, which is usually sufficient. Also after running the gel remove it immediately and keep in the ice cold transfer buffer (TB), the methanol in the TB will prevent diffusion of the proteins within the gel and the gel will expand a bit, after which (around 10 min) you can place the membrane on it. As others said, while transferring keep the unit in cold and try to do the same while running.
We use the following buffer:
6.06 g Tris.base
28.8 Glycine
2.5 mL 10% SDS solution
500 mL Methanol
Make up to 2 L with pure water.
Blotting voltage depends op protein size:
Small proteins: 60 min, 65V
Intermediate (40-100 kD): 70 min, 70V
Large (90+ kD): 90 min, 85V
Hope this helps! Ed Eringa.
Hi thank you all for your reply i really appreciate it. I use the transblot machine, i run cold water longside it and put ice on top of the tubing. The protein i am looking for is around 13kda. I will post a picture in the morning as soon as i can get my camera to work.
Dear colleague
I think your problem relates to your Western blot prior step “analyzing method such as SDS-PAGE”. For testing it, you can stain your SDS-PAGE gel. If your mentioned problem exists in this process, especially for low molecular proteins, you can fix this problem by using Gradient gels.
But if the bands were sharp at stained gel, I think you should fasten well your sandwich and finally, it is better you use methanol in your blotting buffer which prevents swelling the gel from heating.
Kind Regards,
The composition of our transfer buffer is a little bit different:
5.82g Tris.base
2.93g Glycine
0.2g SDS
100mL Methanol
Make up to 1L with pure water.
Blotting voltage is 90V, time depends on protein size. It always works well. And you must pay attention to the temperature. We pre-cold the buffer before wet tranfer and put the Chamber on ice.
Good Luck!
Hi Ayah, I think 100v for 60 minutes is too much especially if you are looking for 13kda protein. Try transfering the gels at 100v for 25 minutes and see if that helps. One more thing is try running the gels in a walk in cold room, as too much heat is generated during wet transfer.
Towbin transfer buffer:
6.05 g Tris-base
28.8 g glycine
400 ml methanol
Final volume 2 liters.
Chill Towbin buffer in freezer for 1-2 hrs. Also freeze ice block insert made for wet transfer unit.
Transfer at 100 V 1 - 1 1/2 hours using the cold buffer/ice insert.
In my experience the bands will smear if the blot get too warm/hot. An alternative to the cold buffer/ice insert is to run the blot at 30-40 V for several hours as others have suggested. Longer times are needed for large proteins. Also I don't know what type of membrane you are using but I find PVDF with a small pore size to be superior.
Good Luck!
Hello Ayah!
It is extremely difficult to get such a small MW protein band to be sharp. You can try running a higher percentage of gel (at least 10%) or a gradient. Also, stop running the gel when the dye front is still on the gel (~1cm for a mini gel or 3-4cm for a larger gel). I also agree with the above comments that say to transfer at a lower V. You should not have a problem with getting the protein out of the gel and it should only take 1-2 hours considering the small MW of the protein. PVDF would also be better (0.2um pore size is perfect!). One more simple tip that you might be overlooking. Make sure not to move the transfer membrane (PVDF or nitrocellulose) after it comes in contact with the gel. You can sometimes get some of the protein to transfer onto the membrane and you see what looks like a smudged band.
I got clear bands on a mini gel for a 16kDa protein by this protocol and they were clean and crisp!
Best Wishes!
attached is a link to my western as the forum would not allow me to attach it to this thread.
I thank you all for your replies, I will try and run my blot at a lower voltage and with the new composition. At the moment i am using PVDF and the blot apparatus is already fixed in a lab, I will have difficulty moving it into the cold room i think. I do run cold water alongside it, and place ice into the side box with tubes.
Is there anything else you would reccomend?
Thank you again
https://www.dropbox.com/s/zqaz69f15vqjbja/photo.JPG
I agree with Melissa Dobson for all his points viz, higher percent of gel (10-12%), run time, 0.2um PVDF membrane, about running voltage, I used 0.8mA/cm square for semi dry transfer and for wet I used 200mA for 2 hours.
Hi Ayah, from your blot pic it seems to be complicated, and I think there should be at least 2 problems. One would be that your gel is not homogenously mixed before casting it. It seems as if the gel is too dense in one side. Second could be non specific antibodies. So, to trace the problem try a suitable dye to stain the gel (coomassie blue) and membrane (Ponceau red) before and after transfer, respectively, and see the pattern.
As additional tip, since u r interested in smaller protein, completely avoid SDS (keep methanol at 20%) in TB (SDS will prevent binding of protein to the membrane), and use membrane with 0,2 micro m or less.
I think running cold water and placing ice is usually sufficient, the aim is not to let the unit heat up which may increase the transfer voltage and further the heat up.
Hello Ayah!
I usually use this TB
3.03g TRIS
14.4g Glycine
200ml MeOH
and make up to 1L with H2OmiliQ
I had troubles with this TB several months ago, one of them was similar that you have, and when I discarded the idea to use SDS in the TB, the transfer worked properly. The other trouble was with the pH. in order to get the right pH, I adjusted using either acid or base according with it needed, however once of the TB is cooled, I saw a kind of precipitation, so I decided to work neither adjust the pH and the addition of SDS. Now the transfer works properly. I hope that help you.
Good luck!
Hi Ayah!
I saw this smearing when I accidentally added too much SDS to the buffer. Actually, in most cases you do not need SDS at all. Just omit it! Another cause could be the heating during the transfer which detaches the membrane from the gel therefore proteins "swim" freely. Do the transfer in a cold room and/or use cooling blocks. Also make sure during the assembly of the blot that your membrane firmly adheres to the gel (press it to the gel by a glass rod or a test tube) and doesn't move during handling.
Good luck!
Peter
For more sharp bands you can try to run the transfer at low voltage, overnight at 4°C. Usually the blot is much more sharp after that. Also always keep ice around the box during the process. In my experience a quick transfer is associated with low actual protein on the transfer paper due to some that is ejected out in the transfer buffer.
Hi Ayah,
First, use electrotransfer buffer without SDS. You can use pre-stained standard proteins to make sure of your electrophoresis and electrotransfer ! If needed, replace and use from time to time a freshely prepared electrophoresis and electrotransfer buffers. Do you activate your PVDF membrane with methanol prior use?
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