Have you tried to add 20% of MeOH to the transfer buffer?

Also, some interesting data: https://www.lifetechnologies.com/be/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/western-blot-transfer-methods.html

Hi Baranya,

First of all, what's the expected kDa size?

Did you manage to see it on the gel using Coomassie blue?

Usually in our lab we use PVDF, as nitrocellulose have the tendency to burn-through, as in the protein transfers through the membrane, and on to the machine or filter paper.

Also when it comes to transfers, the main variable is the amps, not voltage. For our standard 7x6 cm PVDF/NC membranes we use 45mA for 2 hours. You should consider tweaking the amps and the time which the protein is being transferred so the protein doesn't burn through the membrane. Otherwise, it's best to try PVDF for optimal transfers. 

Hope this was useful!

Hi Baranya,

usually I use  14-15% acrylamide SDS-PAGE gels for low molecular weight proteins (12-20 kDa). I use a nitrocellulose membrane 0.22 and add 20% MeOH to the transfer buffer. For a standard transfer phase, I use 100V for 1 hour. The transfer is carried out in ice.

Hi Baranya,

In our lab we detect histones (around 10-15 kDa) in 0,22 nitrocellulose membranes. We run the gel for 1h30 at 15mA (per gel) and transfer in a semi-dry apparatus for 25 min at 16V and 200 mA with 20% meOH tranfer buffer.

Hope this is useful!

Hi Baranya,

this is unusual as normally, there are more problems with transferring high MW proteins than low MW proteins. What type of gel are you using? It is important for low MW proteins to let the gel run slow and to keep an eye on it so the proteins don't move out of the gel at the lower end.

I am usually using 0,22 nictrocellulose membrane and transfer for 30 minutes at 15mA (semidry transfer), and I let the gel, membrane and blotting paper soak in transfer buffer (with 20% MeOH) for about 15-20 minutes before the transfer. This works really well and gives me even amounts of low to high MW proteins (confirmed by Ponceau S staining.)

I hope this helps.

Hi Baranya,

it you perform wet transfer it might help to put a bit more pressure on your gel/membrane sandwich by adding more filter paper, and indeed as already suggested by others, make sure to use methanol and soak gel and membrane for 15min before transfer. Next to this, are you sure you have enough protein to see this using your stain? Maybe you could try a more sensitive stain, or a reversible stain so then you can continue with the western blot after you have completed a good transfer.

all the best with your blots

Marit

Thank you for all your suggestions

Marit Reitsma...

Thank you for your suggestion .i have used 55μg protein sample concentration. still i face the same issue.only high molecular proteins are staining and i couldnot see staining for low molecualr weight proteins.

55ug should indeed be easily visible with most protein stains. Did you stain your gel after transfer to see if the protein is still there? If its not on your gel and not on your membrane then it's probably transfered to the back of your membrane or already in the filter paper behind your membrane. If it is still on your gel I would first go for a "stronger" transfer by doing a longer transfer with higher voltage. If nothing works then I'd try different membranes.

Shih-Liang Lee,

Thank you for your suggestions

Expected kDa size from 15 to 30kDa

Yes i will do try and check

Thank you Marit Reitsma.

 Jennifer Pérez-Boza ,

yes ,i am  using 20% methanol to the transfer.

Thank you

Try reducing current and time. May be low MW proteins are crossing the membrane