Did you try developing the blot with antibody? Although transfer is not efficient, there must be enough proteins transferred into the PVDF (did you see the protein ladder?). In our lab wet blotting works great with 30 V for 2 h.

In addition, not all or complete proteins on the gel will be transferred to the membrane, as the membrane has a defined capacity. So we do see proteins on the gel (with coomassie stain) even after the complete transfer of protein ladder into the membrane.

In fact you will never transfert all the protein from the acrylamide gel onto the membrane, in particular protein that are cherged. Nevertheless since you used PVDF immobilon membranes you can increase the time of transfert because protein are trapped onto the membrane. This iw not amenable when you use nitrocellulose membranes. An other fact, when you say that you trnasfert under 30V what is the amperage during this transfert, we usually transfert under constant amperage (intesity) and this amperagae is given by the area of your acrylamide gel. An other remark the time required for the transfert will also depends on the percentage of acrylamide gel the higher the percentage the higher the intensity of the time needed to transfert.

Good luck

Hi Revathi, starting with sds page you use somewhere around 15 ug of total protein and that is way too much already. if your required protein is like 1% of this total you have like 150ng of protein. Even if your transfer is highly inefficient i.e less than 10% (like 10ng of your protein) still you will be able to detect your protein with antibodies as they are highly specific to pick out ng of your protein. so i second what the two gentlemen said above to run a western blot for your protein. unless your protein is degraded or you have some issue with antibodies the amount transfered is always more than sufficient to detect using western. 

Hope that helps

As Ananda said, you should try blotting your membranes. In most cases you will not get all the protein transferred (depends on how much protein you are trying to transfer and what the membrane binding capacity is). Perhaps you conditions are good enought o detect the protein and further troubleshooting is not necessary.

I always transfer for 1.5h at 120V. If your transfer gets too hot under these conditions, you can transfer in the cold room and include a stir bar in the tank to mix the buffer (to avoid uneven transfer). I found that this transfer is very efficient for most proteins. If you are still getting low efficiency, you can play around with methanol concentration (assuming you are using the Towbin buffer) - sometimes methanol can cause proteins to precipitate in the gel and not transfer efficiently. Also, you can try including SDS in the transfer buffer - SDS will promote elution from the gel, but as it inhibits binding of proteins to the membrane it can still be a problem. On the other hand, SDS will increase conductivity and generate more heat, which can change the antigenicity of some proteins, making them harder to detect with antibodies (or increasing non-specific bands).

In the end, staining a gel after transfer will always give you some proteins, so I think the question you should rather answer is whether you can comfortably detect your protein of interest and only troubleshoot transfer if that's not the case.

If you want to get some help, you will have to provide more details of your protocol. Did you stain your membrane with Ponceau stain after transfer? "Voltage" is not the term you should use to describe protein transfer. What is the amperage that you used? For how long? What is the Mw of your protein? Gel percentage? etc

Some labs use the same transfer buffer many times. If this is true in your lab, try fresh buffer each time. Depleted transfer buffer not only has reduced buffering capacity (or even the wrong pH) but also loses methanol from evaporation on each run. 

Hi Revathi,

I agree with Sereno and Mikhail that you should alway use Amperage when transferring. My lab uses 350mA. It is quite high and therefore gets hot, so we have to change the ice in the middle of the run, but it is quick.

The time required for complete transfer will depend on a number of factors: 

Gel thickness 0.75mm gels will completely transfer much faster than 1.5mm

Gel percentage; the higher the percentage the longer it will take

Protein size; proteins in the bottom of your gel will transfer out faster.

If you are using a gradient gel, the all sizes of protein will migrate out of the gel pretty much at the same time due to the protein size/gel %.

If you tell us more about the conditions (your protein size/ gel percentage and thickness) then we can provide more specific help.

Good luck,

Jacob

ya, my protein size is 60 Kda and  we used 10% gel and i casted 1.0mm thick gel

Efficiency of transfer is determined by many factors: I would certainly western blot the membrane initially to confirm the presence/absence of your protein (also use a positive control if you have one for WB).

It very much depends on the nature of your protein (size, hydrophobicity), membrane used, transfer buffer used (the inclusion of methanol, SDS etc), time of transfer/gel percentage. We have tried a number of different transfer buffers with PVDF membrane and found a simple Tris/glycine mix gave the best results: this worked well for proteins of 25kDa all the way up to 300kDa+. Without more info on your buffer composition and conditions it is difficult to give advice. Alot of people use SDS/Methanol in transfer buffer but this is very often not necessary and in some cases will give rise to poor transfer: you just have to test combinations: every protein is different! Methanol causes the gel to shrink therefore potentially making it difficult for larger proteins to be eluted, it also removes SDS which can lead to ppt. SDS aids in the solubilisation of proteins that are prone to ppt in the gel. I would also check for loss due to poor binding by transferring in the presence of a double membrane: it is possible that protein is not retained by the PVDF. Remember PVDF needs pre-soaking in methonol prior to use

I would suggest you start by looking at the literature: Abcam produce an info sheet for the beginner in their protocol section.

good luck