l used both. Although my colleagues prefers tank transfer I think semidry is better for efficient transfer in short duration and saves buffer and hazardous methanol. care must be taken while setting the voltage(and time) according to the size of the protein.

but still don't change the winning team, if u feel tank system makes better,then stick to that.

Actually I find the tank system is prone to getting bubbles in the transfer. Very hard to weed out all the bubbles no matter how much you role a testtube over your gel. Is there less bubbles with the semidry?

i use semi-dry all the time and get good protein transfer. However, I discovered that 7.5% separating gel for SDS-PAGE, though a bit wobbly gives good protein transfer. in addition make sure the layers of filter papers sandwiching the gel and nitrocellulose paper are adequately soaked to avoid drying out, run on mA for 1 h max.

Hi Dayan,

It depends on the size of your protein. Larger ones - go with tank. Smaller ones - go with semi-dry. Larger proteins are difficult to be released from the gel and therefore need more transfer time and in this case the tank system is better because the temperature is kept lower. For a really complete guide on WB, refer to the Biorad SDS-PAGE and WB guides (biorad.com). They are extremely detailed.

Good luck!

I personally prefer semi-dry, especially with one of these fast transfer buffers (be it Lonza or Pierce). The transfer takes only 10 min! Like it was mentioned before, it is easier to avoid bubbles with semi-dry. Setting up the sandwich is really hassle free. We do all transfers using fast semi-dry, however in our lab we don't work with anything bigger than 130kDa. I know that semi-dry is still OK for 200kDa, but anything bigger probably should go via tank system (even though you can play a little bit with adding some SDS to your semi-dry buffer to increase large protein recovery). If it comes to membranes, we routinely use PVDF. It has better physical properties than nitrocellulose (less prone to tearing) and can be quite easily stripped and re-probed. Millipore and Pierce have excellent western blotting handbooks, with all possible variations and trouble-shooting.

regarding buffers, we use normal sds gel running Buffett plus methanol to make the paper semi-dry, it us working for us without any special readymade buffers. we run about 30min.

We only use semi-dry for the reason that it's much faster. Indeed it's less efficient with very large proteins (>150-200 kDa), but you can get them through by increasing the time at high current (2h is usually sufficient). But make sure that there is enough buffer, by putting 1 or 2 more layers of soaked paper in your sandwich.

I have used both systems and I prefer semi-dry to wet transfer.

Currently, I use 4-12% gradient gels for SDS-Page with 1X MES buffer and semi-dry transfer with 1X Transfer Buffer (diluted in methanol from 10X Transfer Buffer= 25 mM Tris-base and 2M glycine) at 80 mA for about 1 hour. the proteins that we are interested in are between 40-130 kDa.

We used both PVDF and nitrocellulose membranes and are happy with the outcome. As mentioned above, we have not used the semi-dry system for any protein bigger than 150 kDa and the size of your protein is something that you need to consider.

Regards to bubbles - less likely to get bubbles in semi-dry relative to wet-transfer.

Good luck!

I always used semi-dry...simpliest to use and very efficient. I work on proteins from 3kDa to 280kDa!! But i am using discontinuous buffers.I have attached 2 files to explain how you can proceed.

Here is the first file

And the second one

Hi, I used both and have good results in general. Wet-transfer might have another advantages: it allows a more homogenous transfer of proteins (higher reproducibility - especially for big proteins). I was quantifying proteins by Western blot, for this reason I switched to wet-transfer. Concerning the membranes:the PVDF membrane has the advantage to retain the proteins as they can get through the nitrocellulose if you transfer too long.

I have also used both and prefer the semi-dry version these days.

If you have any chance of getting a hand on a iBlot system from Invitrogen, get it. With it you can blot two mini gels at the same time (and comparable costs) in just 7 minutes.

I used semi-dry for my smaller protein which is >30kda and it worked fine. As everyone else said for larger protein you should go for wet system. Good luck!

I would use the wet system but if your expected protein's size is less than 40 kDa you can use semi dry.

Semi-dry is preferable.

PVDF or nitrocellulose is a choice you should do depending of the size of the protein + antibody establishment.

The choice of semi dry or wet tank is to my mind more a question of material and protein weight. I have more reproducible data set using wet tank

PVDF membranes worked fine with me, also for high MW proteins, too. If you plan to use PVDF as a membrane, just do not forget to condition your membrane before blotting. The PVDF surface in hydrophobic so it won't get wet directly in buffers. Briefly you have to wet it first with MeOH (soak it in 100%MeOH) for 10 mins and then soak it to your transfer buffer (Towbin Buffer is good ) at least 15 mins before casting your sandwich.

Both SemiDry and Wet blotting works fine, it mostly depends on which one is more easy to cast for you. I would prefer semiDry, since easier for me to cast.

The transfer efficiency depends on many factors, like time, voltage, buffer (MeOH and SDS concentrations) . So whichever method you use, you would need a good protocol.

We have both set-up here in our lab. We usually use the semi-dry transfer system for small gels (for 1mm thickness gels). The transfer is very efficient for smaller size proteins. This can be seen by staining the post-transfer gels. As for big gels (or thicker gels), we prefer wet transfer system. Among other disadvantages of wet systems are it requires more buffer and it need cooling system due to the heat generated.

Semi dry method is better.It takes less time and % transfer is more.

We always use PVDF. For proteins ~150 kDa and up I use the standard tank transfer. For proteins lower in MW than 150 I use semi-dry. For smaller proteins only a 10-20 minute transfer time is needed with this method. Semi-dry will transfer 150 and 250 kDa proteins, but a lot will be left in the gel.

I used PVDF membrane with semi-dry method. It worked well with my proteins.

i Will prefer using Semidry together with PCDF membrane under 18V, Max mA for an hour.

I used 20V for 30 min

Although I use a modified semi dry procedure...the wet setup is considerably more efficient. As has already been stated semi is better for sub 100kda proteins and wet is good for all proteins. The only draw back for wet is time and consumables. I have a rig from bio rad called the criterion which can transfer well in 30 minutes.

I agree with most answers here. We use both techniques daily. Semi dry is great for its speed and low toxic waste, albeit more expensive. By using semi dry transfers you can have great transfers in 7min (as opposed to 1hr+) with no loss of signal. For big proteins we find the slow wet transfer to be more effective. The quality of semi dry transfers might change with the brand. We use the BioRad TransTurbo blotting device.

I found PVDF membrane better than nitrocellulose because it absorbs more protein; hence better protein detection and I also found semi-dry blotting quicker than wet blotting.

I think just for faster. I have done the both methods with same protein... The result is similar but semi is faster.

Thanks for all the feedback everyone. It was so much appreciated

Despite of all arguments for the semidry:

1. tank blotting is much more sensitive. Why: when you start the transfer, an isotachphoresis starts to concentrate the proteins in the gel, but they are still non on the membrane. This takes some times due to the small pH-differences between the gel and the transfer buffer.

2. The workflow is much better with a tank blot. You can prepare and run the gels until afternoon. Then you can start the overnight tank blot (at 15 °C, not lower, SDS will crystallize at lower temperatures), come in the lab next morning in the lab, go for e.u. reversible staining, blocking, ... and do the immune reaction.

3. The sensitivity is much higher because of the much more effective transfer of all proteins, including proteins with a high molecular weight.

Standard transfer is a bit longer, but it allows more efficient transfer of protein mixtures containing broad range of proteins, or 20-40 mere peptides (using 4-20% PAG or 15% PAG), that is difficult to achieve by semi-dry approach. The reason - is a high voltage used for the transfer, that promote peptides passing through 0,22 membranes. Another advantage of a standard approach is a partial renaturation of some proteins when the transfer is performed for 3-4 hours with 10% methanol. This can be seen by running comparative Western blots using a reference serum.

However, all depends on your task.

It will be better, if the semi dry method is adopted for better transblot of protein onto NCP or similar matrices.

Yes, this is true.AMJana

In our hands wet western blot transfer works excellent and very much reproducible. Stay wet :)