Hi Soumik,

Can you provide your transferring conditions and the buffer composition? It's hard to comment without those details.

Usually considerable amount of larger proteins remains in the gel after the transferring. I would suggest you to stain the membrane with Ponceau S and see whether there are proteins on the membrane. If you still didn’t get the proteins you have to check your transfer buffer.

Adding 0.05% SDS into the transfer buffer can increase the transferring efficiency (Note: Do not change the methanol concentration)

Since you are getting the same results from both wet and the semi dry transferring, I assume the problem must be with your buffer (assuming the transfer conditions are correct)

Good Luck,

Susara

Hi,

Can i ask you some few questions(1)What is the size of your sample protein?Note that larger protein needs more time to transfer(2)Please i need to clarify that what condition did you use such voltage,current and duration of transfer?(3)Did you use new or re-use transfer buffer(4)Did you equilibrate your PVDF membrane before use and avoid bubbles in your transfer cassette?If you can sincerely answer these questions, this can help you solve your problem.

NB-if the MW of your protein is large,it is worthy to note that you add some SDS in the transfer buffer like 0.025-0.5% because SDS helps in the elution of your protein from the gel and decrease binding efficiency to PVDF membrane while methanol plays and opposite function to SDS by removing SDS from protein and improving the binding efficiency to PVDF membrane.

I hope this will help.

Proteins size; 1 band around 100kd, 2 bands around 45 kd (major concern)

semidry: Transfer Buffer: 25mM Tris, 150mM Glycine, 20% Methanol

condition: Constant current 50mA for 1 hr

Wet: Transfer Buffer: 48mM Tris, 39mM Glycine, 20% Methanol

condition: Constant voltage 100V for 1 hr

Hi Soumik,

Though the semi dry transferring is easy, still the wet transferring method is the golden standard for proper transferring.

I see your transfer buffer has some differences. I would suggest you to try this buffer composition.

For 1X Transfer buffer: 25 mM Tris, 192 mM Glycine, 20% (v/v) methanol, pH ~8.3. (Cell Signaling Technology Cat #12539) This has never disappointed me. You can use the same buffer for the both wet and semi-dry transferring methods.

Further, use following conditions for the wet transferring: 300mA constant current for 2h at 4C (you can keep the apparatus in a cold room or cover with ice).

Usually the voltage should be above 120V for a proper transferring. Once you set the constant current (300mA) you would see the voltage begins from 190V and drops down to 120-110V.

You must get the bands on your membrane. Do not forget to stain the membrane with Ponceau S before you block.

Good Luck..!

Susara

Hi Soumik,

U had used PVDF member... I m right. If so, did you pre activate ur PVDF membrane with methanol. As this step is missing in your question.

I will suggest you to include this step or use nitrocellulose member and check, protein is transfer or not.

Secondly, if your are transferring a protein with low molecular weight their may be changes that your low molecule weight protein run away from the member. But in high molecular weight protein at your mention details will never run out the member.

So, please check in ponceau Stain and confirm that protein has transferred

Thank you, everyone, now the problem is solved. I have used 0.05% SDS in buffer (both Wet and semi-dry; 48mM Tris, 39mM Glycine, 20% Methanol).

Still, the transfer efficiency is 50-70%, but it will serve my purpose. Thank you all for your suggestions.