Hello Isma.

There are many factors that can be the cause of your problem.

- Are you performing a semi-dry or a wet transfer in a tank? For a semi-dry transfer you should run at 0.8 mA/cm2 (-> this is calculated from the dimensions of your gel) for 1-2 h.

- pH of the buffers used

- Do you wet your membrane with alcohol, before blotting?

-...

The handbook below might be helpful:

http://web.mnstate.edu/provost/ProteinBlottingBook.pdf

Best regards

hi marcia , i am carrying out wet transfer and not adjusting ph of transfer buffer , have used methanol to wet membrane for 15 sec before blotting

You should try cold transfer for better results. 

Best,

Hamadi. 

Base on your description, neither 15kv overnight nor 60kv 1 hour did not transfer your protein out from your gel. And how about your ladder, all have been transfer to the PVDF membrane or just the lanes at small MW? And what is the MW of your protein? And also I think you could try high voltage (cold transfer with ice box), like 100kv for 1H or reduce the filter paper your used in sandwich. Then stain both gel and PVDF to check where is your protein, on the gel or PVDF or pass all. 

Best regards

Protein ladder is transfered completely but protein bands are very light as visualized by ponceu staining. After gel staining to confirm transfer after western blot i can visualize protein bands clearly indicating incomplete transfer.

2hours at 200mA constant current.. (this gives around 100V which reduces after a while) This usually works for us.. To avoid heating you can use either a cold pack or do it in cold room.. But we normally do it at room temperature and it works. Its also possible that you see faint bands of proteins using ponceau due to several reasons.. Either you are loading too less but you see them on the gel as you said, so could be that the ponceau staining is done for a lesser time.. If you see bands after developing western then its enough..  As its not unusual that some proteins stay behind on your gel.. Most likely, increasing the time and current will help..

What is the size of your protein?

320 mA for 1,5 hr always works for us without any proteins remaining on the acrylamide gel. We perform the transfer process in a cold room and we use a magnet and a magnetic stirrer. I would strongly suggest that you equilibrate your PVDF with transfer buffer after soaking it in methanol for 1-5 minutes. The same applies for the acrylamide gel. This a good practice in order to ensure a well assembled "sandwich" of all the parts involved in wet transfer (namely gel, membrane, Whatman papers, sponges). Is your transfer buffer hand-made? Does it contain SDS? Hope everything works out eventually..(I have attached some useful bulletins)

Hi there,

Many good answers already. In my case, ON 35mA or 90min 350mA wet transfer always gives perfect transfer (no left over in the gel) irrespective of the protein size. Maybe I can give you some more ideas:

-Did you try Nitrocellulose membrane? In my experience, PVDFs are more sensitive and delicate for Ponceau S, stripping and re-incubations. They also need 5min methanol incubation for activation prior transfer, and after Ponceau S.

-Do you have the chance to use company produced buffers? Nothing against home made, but this can rule out possible intereference with pH, old reagents, etc.

-How is your sample? If it's a lysate with RIPA buffer, or contains any clotting molecule this could also interfere in your transfer. Do you use reducing conditions? Is your SDS-buffer ok?

I hope this helps!

best!

hi there... 

PVDF membrane is positively charges but needs to be activated in 100%methnol for atleast 30mins prior to make cassette. In order to increase transfer efficiency of you protiens you can increase the methnol concentration upto 20% and glycine same as in the running buffer as glycine acts as a bridge for protien migration. Choose to wet transfer for overnight at 15-20V however in Our lab 2hrs at 75-90V does the job. Also use a 0.45 micron membrane unless you are targeting very low molecular weight protiens i.e.

Hi dear,

If you have a larger sized protein then try 60V for ~2 1/2hr  to 3hr instead of 1hr. Also keep the apparatus in cold environment (4 degree C). Also keep the transfer buffer in 4 degree C prior to use.

Best wishes.