Did you stain your gel *after* ther transfer in coomassie to see if there is any protein left? If this is the case, you could use a longer transfer time. Big protein are sometimes not as easy to transfer as smaller.

The other thing: You are using gels with a quite high percentage for the big proteins. Have you tried using 8% gels to see if they work better?

I usually find that wet transfer overnight is really good. 25V overnight in cold room with a stirrer (magnetic) at the base of the transfer chamber to allow for avoid the development of a temperature gradient that might affect efficiency of transfer of some proteins.

I didn't do a commassie stain however I am sure that there are proteins left on the gels cuz the high mw ladder failed to be transfered onto the membrane and left on the gel. We use 10% gel to get a decent seperation for big proteins and at the meantime keep the smaller mw protein not running out the gel. BTW, What is the mw of your target proteins when you do the wet transfer overnight.

It is usually around 60-90kDa. But after the O/N transfer I can see all the bands nicely transferred onto the membrane. I did this with a home made 10% gel too and have been successful with both nitrocellulose and PVDF membranes

I mean the colored ladder bands.. try the O/N and update us

I will try. Thank you! BTW, what buffer you use for the wet transfer?

We used 7-8% gels for a range of 35-90 kDa, a collegue got over 120 with it.

I am not sure if I understand you right, but you say your high MW marker is not transferred on the blot and stays on the gel?

If this is the case stain the blotted gel and you will see it. I routinely do this, throw it into the staining solution and have a short look on it. Makes absolutely no work and your proteins are not going to disappear. I usually also stain the membrane with Poinceau red.

Their is two main idea:

-> amount of methanol / SDS which is wrong in transfer buffer

-> composition of acrylamide gel is wrong

have a look on this

http://www.millipore.com/techpublications/tech1/tp001en00

Hi Zezhi,

I have a few suggestions for you, but should start by saying it might need a bit of optimisation to get both low and high MW proteins to transfer efficiently in the same blot (depending on the nature of the proteins).

When you say: "sometimes get good results, sometimes the bands were swirling or disappear (not all of them but part of the gel)", this suggests poor contact and/or bubbles between the gel and the membrane, and it's very frustrating when it happens! Try using at least one more piece of filter paper each side of the transfer sandwich when using wet transfer to get a better contact, and be sure to roll out any bubbles carefully. This should help you get more consistent results.

If your high MW markers are not transferring, you definitely need to do a longer transfer. I use constant current for transfers; and would try 250 mA for 90 min to begin with, then up the time if transfer is incomplete.

Also, PVDF membrane can capture some proteins much more efficiently than nitrocellulose. If your markers are transferring OK but one of your bands is still weak, you could try this. I've had a lot of success with PVDF for some proteins which will hardly stick to nitrocellulose at all.

There are other "tweaks" you can try, particularly if you have hydrophobic proteins, but see if the advice you've got from the comments so far helps...

Good luck!

Mark

There are several ways to optimise the traditional wet transfer as some have already mentioned. One is to decrease the % of methanol, say, to 10%. A lower % gel may also help. Another thing is, you can think about using a thinner gel? I presume that you are using 1mm, so if you do not require to load large volumes of samples into the wells, try using a 7.5mm gel instead. Some of our colleagues put 0.1% SDS into the transfer buffer when transferring large proteins, but I personally do not think it enhances the transfer.

As for the BioRad semi-dry transfer, unless you are using their pre-cast gels and membranes, from our experiences, you can never get good transfers for anything larger than 10k. Increasing the transfer time does not help as you may burn your both of your membrane and gels.

Hi Yuan:

This is my best recommendation for your next WB. Because you are using nitrocellulose membrane, I recommend you to treat the NC membrane in 20 % methanol solution for about 30 min at RT. After you finish the electrophoretic separation of your proteins you should wash the SDS-gel by using ddH2O. Besides, I recommend you to transferring the proteins using 30 mA (constant mA) overnight with cooling (4°C). I am pretty sure you will get the results you are expecting for.

Best Regards,

Arnubio.

Hello Zhezhi

I hope the troubleshooting is working better after the great advice given by several colleagues. I used to work do western blots of a subunit that was 565kDa... it was a protein monster that one! I learnt that big proteins appreciate the space to migrate in gels and to get out of them during transfer. First of all I would recommend to make 1.5mm thick gels rather than the usual 1mm. I think 10% acrylamide/bisacrylamide is fine for a 135kDa protein but I was wondering what was the ratio between the two in the recipe you are making... for me the ratio that worked best was 37.5:1 which I've later found out is not very usual (this is just empiric observation but I guess it's all about space for proteins to move within the gel and to get out of it). Finally, my best transfers were overnight (about 14 hours) at 25V and at 4ºC, as Dr. El-Zeiry suggested in her post. I also added 0.01% Tween-20 to the transfer buffer that may help big proteins to transfer better (but it makes more bubbles).

A word of caution: when you set up the conditions for the transfer of big proteins, you may lose the smaller proteins since they get through nitrocellulose. As Dr. Thompson suggested in his post, I would rather use PVDF. There is even a version (I think) of a PVDF with a smaller pore size than the usual that may help to retain them. Good luck!

Proteins > 75kDa is often poorly transferred with the semi-dry system. The easiest way to get around this problem is to use PVDF membranes (small proteins will stick) and longer transfer times (for mini-gels I use 120mA for 1h).

Hope this will help.

Thank you all! For all those suggestions! So, it seems like wet-transfer overnight in coldroom at 25V is the solution that I could use right now. One thing I am worry about is, will this overnight transfer make my 27 kDa band disappear though larger bands like 135 kDa stays?

Hello Zezhi,

Here are some of my suggestions,

1) Use ice cold transfer buffer and do your transfer in cold condition.

2) Use 20% methanol in your transfer buffer.

3) Apply 300mA for 1 to 1 1/2 hour and try to maintain volts below 100V to avoid heating up of your transfer system.

4) Use ice pack while transfer and Do not hesitate to change it in the middle.

I usually follow these steps and never faced any problems with transfer.