I used  this system - it works really good. There are different protocols for different protein sizes in a Turbo manual - if you choose the appropriate protocol that will be fine. There is also a western blot manual on the web-page (http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_2895.pdf) - there're also many useful tips in it.

There is a general-purpose Turbo protocol which runs 7 minutes - in this case high MW proteins probably will not be transferred. I used ready-to-use sandwiches which are already equlibrated  in transfer buffer but my colleagues used home-made ones and replaced methanol with ethanol in transfer buffer - it makes transfer slower (for example, you have to run Turbo protocol like 20 minutes instead of 7) but it's cheaper and safer to use. 

If you transfer low or medium MW proteins it's better to use PVDF membranes - they have smaller pore diameter and in general are of a better quality.

Hello Edward,

I did both dry and wet transfer a long time ago using our lab-made gels, NC membranes and basically what I found is that the dry transfer does not transfer as much protein as the wet transfer. It takes more practice to get used to (and do well) for dry transfer since you cannot put too much buffer in the transfer unit. Even for dry transfer, you still need to pre-wet all the materials for the assembly. 

If possible, I would suggest you get a demo unit from bio-rad and compare side by side which method works better in your hand. I think different people do have different preference when it comes to transfer. 

Regarding the voltage to use, better to follow the recommendations of the manufacturer. 

Best luck!

Zoe

Looked at this Trans-blot Turbo but didn't buy it. 

a. Do you pre-wet your filter paper and nitrocellulose membrane?

              yes, although the ones they are selling come pre soaked and individually                           wrapped. 

b. How long is the transfer (in minutes) and what is the voltage (and other parameters) used?

              Depends which machine you buy and size of your protein, but a good starting                 point is 75mA  / 1 hour. I think this turbo is made to go much quicker, thus                       their consumables being so critical for use. 

c. What are the things to consider? Bad experiences?

            I prefer dry, no problems with ugly air bubbles. 

Comparing it with wet transfer, how good is the overall transfer of proteins to the membrane using this dry transfer?

              You should be considering the size of your protein when optimising transfer,                      both wet and dry are established, reliable techniques. Also consider alternative               membranes like PVDF.

In Western blotting, what is your experience with Wet vs Dry Transfer Systems?

             See c. :)

Hi

I have been using wet transfer method for long time and I got good results for all kind of proteins. According to your situation I suggest you to use wet method according to following protocol

a. Do you pre-wet your filter paper and nitrocellulose membrane?

Yes I pre-wet filter paper in transfer buffer ( with 20%methanol) and PVDF membrane first in 100%methanol for 30 sec and then in transfer buffer.

b. How long is the transfer (in minutes) and what is the voltage (and other parameters) used?

I used transfer time of 1 h at 80 mA for around 90 KDa protein that works well. 

c. What are the things to consider? Bad experiences?

things are to consider the quality of your gel (if homemade) and air bubbles during transfer and try to transfer at low temperature.

All above I used with home made gel.

Wish you good luck.

For more difficult proteins (low amounts in a lysate, of hight molecular weight) I use wet transfer (now with pre-cast gels, but earlier with hand-made gels). Now we often use Turbo Transblot from Bio-Rad - also pre-cast gels but membranes sometimes are from ready Bio-Rad sandwiches and sometimes we cut them by ourselves (yes, we wet them according to themanufacturers protocol). We have never used hand-made gel for the Turbo system. In Turbo sysytem you have programmed ready protocols, we usually used "Mixed Mw" - it works in many cases.

I used  this system - it works really good. There are different protocols for different protein sizes in a Turbo manual - if you choose the appropriate protocol that will be fine. There is also a western blot manual on the web-page (http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_2895.pdf) - there're also many useful tips in it.

There is a general-purpose Turbo protocol which runs 7 minutes - in this case high MW proteins probably will not be transferred. I used ready-to-use sandwiches which are already equlibrated  in transfer buffer but my colleagues used home-made ones and replaced methanol with ethanol in transfer buffer - it makes transfer slower (for example, you have to run Turbo protocol like 20 minutes instead of 7) but it's cheaper and safer to use. 

If you transfer low or medium MW proteins it's better to use PVDF membranes - they have smaller pore diameter and in general are of a better quality.

Hello Zoe,

I did both dry and wet transfer a long time ago using our lab-made gels, NC membranes and basically what I found is that the dry transfer does not transfer as much protein as the wet transfer. It takes more practice to get used to (and do well) for dry transfer since you cannot put too much buffer in the transfer unit.

- Did you resort back to wet transfer and cease using dry blot?

Even for dry transfer, you still need to pre-wet all the materials for the assembly. 

-OK, I got it. Thanks!

If possible, I would suggest you get a demo unit from Bio-rad and compare side by side which method works better in your hand. I think different people do have different preference when it comes to transfer.

- I may not be able to ask a demo unit; we already have it. I will soon find out how good it is for our assays. I just need to know how efficient this is for what I am going to do, because I am used to wet transfer, but dry blot seems very enticing to me.

Regarding the voltage to use, better to follow the recommendations of the manufacturer.

-OK! Thanks

Hi Jennifer,

I appreciate your point-by-point response. :)

Yes, although the ones they are selling come pre soaked and indiually wrapped.

- I think that is specifically for the dry blot. But they also sell nitrocellulose membrane rolls that are dry. which we use for wet transfer. So just soak it in buffer before using, right?

Depends which machine you buy and size of your protein, but a good starting point is 75mA  / 1 hour. I think this turbo is made to go much quicker, thus their consumables being so critical for use.

- That is what I am trying to avoid - using proprietary materials.

I prefer dry, no problems with ugly air bubbles.

- Good to know you like dry transfer.

You should be considering the size of your protein when optimising transfer, both wet and dry are established, reliable techniques. Also consider alternative membranes like PVDF.

- I will consider that. A lot of answers here said that larger proteins are not transferred efficiently. I wonder what sizes are they pointing out.

Thanks,

Edward

Hi Zubair,

Thanks. We have both wet and dry transfer systems. I just want the method with less steps and lesser materials, thus, asking about dry transfer. I also used wet transfer for a long time and it's great.

Thanks,

Edward

Hi Karolina,

For more difficult proteins (low amounts in a lysate, of high molecular weight) I use wet transfer (now with pre-cast gels, but earlier with hand-made gels).

- That's a good information!

Now we often use Turbo Transblot from Bio-Rad - also pre-cast gels but membranes sometimes are from ready Bio-Rad sandwiches and sometimes we cut them by ourselves (yes, we wet them according to themanufacturers protocol).

- I am trying to optimize my assay using home-made materials though. So there's a way to wet the nitrocellulose membrane? Can you send a link?

We have never used hand-made gel for the Turbo system.

- Is there a specific reason why you don't use home-made gels? I make my gels all the time.

In Turbo system you have programmed ready protocols, we usually used "Mixed Mw" - it works in many cases.

- Good to know. Thanks!

Hi Darya,

Thanks for the link and all info! Can you let me know how to equilibrate membrane with transfer buffer? That would be helpful! I will consider switching to PVDF, for now, I am stuck with Nitrocellulose Membrane. I appreciate your answer!

Sincerely,

Edward

Edward,  

For PVDF membranes I  wet them 30 sec in methanol,  rinse with distilled water (2 min) and then with transfer buffer  (15 min). For the turbo transfer is good to buy Bio-Rad's Turbo buffer that is concentrated. Then the equilibration is shorter, I think (now I have holidays and do not remember everything, hopefully). We do not perform turbo blot with our home-made buffers and we have never tried to do that. For sure there is sth special in Bio-Rad's buffers that makes the Turbo transfer works.  It  is good you hire the apparature for some time and try your options. 

Hi Edward,

You just thoroughly soak each part of the sandwich in transfer buffer. Everything is written in detail in this blotting guide - page 27-28.

I have always made my gels myself, with the Hoefer SE260 system. For the large proteins (80-160 kDa) that I am interested in, tank blotting IMHO works better than semidry. I use a 60 V, 1 A regulated power supply (less than 100 $ in an electronics shop, frees up the expensive electrophoresis power supply), the actual current with a Biorad tank blotter is about 200 mA at 40 V.

I always use PVDF, not nitrocellulose, because PVDF is mechanically more stable (never breaks during incubation with antibodies) and has a higher protein binding capacity and affinity (proteins don't slip through the membrane during blotting). PVDF neets to be wetted with methanol (a few seconds), then washed with dd water, then equilibrated with buffer.

For SDS-PAGE gels, I use Dunn's blotting buffer (Anal. Biochem. 157 (1986)144-53), which is cheaper than Towbin's and IMHO works better.