hi,everybody.I'm just starting to do western blot,and so many problems came out.
I just tried to transfer 250kda protein,but failed all the time.So I need some advices and to improve my protocol.
I used 5% separating gel,and 1x transfer buffer (20% methanol),7v for 4 hours.After 4 hours,I found the gel transparent，and PVDF membrane do have the marker ,but fliter paper were yellow,I have no idea why filter paper were yellow.
Waiting for your help,thx so much
Hello,
You already have some good answers. I just want to point out to the fact that, for high weight protein, you might want to decrease the methanol content of the transfer buffer which usually counter acts the SDS thats already there. Having more SDS means you have more negative charge on you proteins which will help in the transfer. Finding the right SDS/Methanol Balance is critical in my opinion.
Good Luck :)
1) You can use TWO membranes stacked one atop of the other, to make sure the proteins being blotted do not pass through.
2) You can reversibly stain ponceau red to ensure the transfer succeeded.
Do you mean the "Yellow" as "Burned"?
If there is heat on filter paper and/or membrane after transfer, I think the current is high.
In my laboratory, we used water-tank transfer machine (not semi-dry transfer) to transfer over-200 kDa protein.
Or in semi-dry condition, you can set up the transfer condition as 200 mA for 4 h rather than 7V.
I agree with So-Jin Kim, for blotting large proteins semi-dry is not really recommended (although it can be done). I have attached a semi-dry blotting protocol of mine. With this I had often success to transfer big proteins. Good luck!
I agree with So-Jim and Christian, semi-dry is better for proteins of 70 - 10 kDa MW (in my short experience), and for your proteins is better use water-tank and also I recommend you use a transfer buffer with 0.05% of SDS and 20% of methanol.
"The efficiency of transfer of high molecular weight antigens may be
enhanced by increasing transfer time and the addition 0.05% SDS to the transfer buffer. Typically, the more hydrophobic a protein is, the more difficult it may be to transfer to solid support membranes. The addition of methanol (up to 20%) to
the transfer buffer has proven to enhance the transfer efficiency of some hydrophobic proteins.. " (INTRODUCTION TO ANTIBODIES, 2º ed, Chemicon International)
Thank you guys,thank you all so much for helping me.And I will change the protocol to try again ~~
Hello,
You already have some good answers. I just want to point out to the fact that, for high weight protein, you might want to decrease the methanol content of the transfer buffer which usually counter acts the SDS thats already there. Having more SDS means you have more negative charge on you proteins which will help in the transfer. Finding the right SDS/Methanol Balance is critical in my opinion.
Good Luck :)
Hi Ma,
Remember to activate PVDF by rinsing it with 100% methanol first. Otherwise, you will not obtain a successful transfer.
As already pointed out, having small concentration of SDS in transfer buffer help with high molecular weight protein electroblotting.
You should also try and get the protein to move to about mid-section of the membrane. Transfer efficiency is good in the middle of the blotting assembly. If the protein is very high on the gel, your transfer efficiency will be poor.
Good luck. Hediye
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