I would try a shorter time, between 60 and 90 minutes, using 70-90 volts. Also, you can do it with no SDS in the transfer buffer, and lowering the methanol to 10%. Keep the buffer cold to avoid heating (putting the transfer tank on ice). I have been doing it like that for years and never have such problem with small proteins. Good luck!!

Are you sure it is 0.1% SDS, which is 10 times higher than what is used in our lab.

I suggest you try 350mA for 1hr10min or 48V overnight while using 0.01% sds

Ok, so from your reply, I have a lead as to what could be going wrong - There was some error in my calculations - I have been adding 0.37g SDS per litre (0.037 %) instead of 0.1%. Actually there are numerous formulations available on the internet. People suggest using SDS in the range 0.01% - 0.1%. Let me try with a fresh buffer containing correct SDS concentration. Thanks for pointing out Siyuan Su!

I usually use 0.8 mA multiplying with the cm2 of the membrane you use. Good Luck ! the procent of SDS is indeed too high !

I would try a shorter time, between 60 and 90 minutes, using 70-90 volts. Also, you can do it with no SDS in the transfer buffer, and lowering the methanol to 10%. Keep the buffer cold to avoid heating (putting the transfer tank on ice). I have been doing it like that for years and never have such problem with small proteins. Good luck!!

Hi:

we use the same aparatus with a .45 um membrane. 90 min in fixed 100mV work just fine to transfer virtually every protein (at least all the full range marker). Our blotting buffer has (24 mM Tris, 192 mM glycine containing 20% methanol). Good luck!

Hi

Very simple rule that I learned during my Ph.D. working with senior people. Using a classic transfer buffer (20% methanol) a protein with less than 100 kDa should transfer in semi-dry conditions at 1 mA/cm2 during 1 hour. Use intensite instead of voltage.

I prefer semi-dry conditions if you have the proper system. Wet transfer is very expensive in terms of buffer.

Hope it helps Good luck. Cheers

Paco

for a 25kd protein you do not need 0.1% SDS.

What I usually do is measure the length and the breadth of NC membrane and then calculate the current in mA as: L * B * 1.5 mA. Transfer the proteins for 1.5 to 2 hours.

Thanks everyone for the answers! I checked the composition of Bjerrum Schafer-Nielsen buffer from various sources and I concluded that there is much ambiguity - some sources say 0.37g while others say 0.037g. I'm still not certain about the correct concentration. But what I could infer from my reading is that for smaller sized proteins (especially in case of NC membrane), it is advisable to go for decreased SDS concentration. For ~25kDa sized protein, less than 0.05% SDS should suffice. So I feel 0.037% that I used in my buffer is appropriate. I think it is the transfer time/power setting that I need to optimize. As suggested in the comments above, I will try the rapid transfer (high intensity / less duration) this time. Will update my findings soon. Thanks again!

I usually use a 7-20% gradient (home brew) 1mm thick gel, and do the transfers in 1x tris-glycine (reciepe from biorad). at 400mA proteins all are transferred in 1 hour in tank, in a semi-dry blotter the transfer is variable. there is no need of sds for such a small protein, infact adding sds to such a small protein will work against you.

you can run transfer your protein either at a voltage of 30V for overnight incubation or 60-80V for 1 hour.if you are transferring at 60 V then you should immerse your transferring system in ice otherwise too much heat will be generated

I'm curious about the different opinions on SDS. Assuming that the samples were prepared and run in a buffer containing SDS, they should already be negatively charged, so is there really a need to have SDS in the transfer buffer for that purpose as well? And if not, what is the function of SDS in the transfer buffer?

SDS serves many purposes: it maintains negative charges on the protein that help them migrate to the positive electrode in your electrophoresis unit, it unfolds the protein from its native conformation promoting better transfer and it helps separate those proteins in your sample that could migrate together due to electrostatic interactions.

Most of those purposes are more relevant to the initial electrophoresis than to the transfer though. Shouldn't the proteins still be negatively charged from running the gel?

I have been using the Bio-Rad Criterion system. I use the one with thw cathode plate. Bio-Rads 10X transfer buffer, (700ml DDH2O, 200ml Methanol, 100ml of 10X transfer stock). Make up the day b4 and chill to 4*C. Pre-incubate fiber pads, filter paper, and completed gels for 30 mins @ 4*C. I run @ 100V for 42 mins. Cheers!

I am not sure if it fits for the situation of the question owner but just a tiny trick, in case you are looking at several proteins of various sizes on a single run: You can put an additional membrane on top of the membrane you added for the transfer. Then, you obtain the bigger proteins on the first membrane and the smaller ones on the second.

0.4 mA, 20-30V, 50 mins

Complete answer is here... from a BioRad guide to transfers with standard Tris-Glycine-MeOH (Towbin) buffer... adapt to your specific equipment:

...............................|..Standard (hrs).........|..High-Intensity (mins)

Trans-Blot tank

• Plate electr........10 V/100 mA, 16 hr....|.50–100 V/700–1,600 mA, 30–60 min
• Wire electrode...30 V/100 mA, 16 hr....|.100–200 V/300–800 mA, 30 min–4 hr

Trans-Blot Plus.....30 V/0.5 A, 16 hr.........|.100 V/1,500 mA, 60 min

Mini Trans-Blot.....30 V/90 mA, 16 hr.......|.100 V/350 mA, 60 min

Criterion blotter

• Plate electr........10 V/50–80mA, 16 hr..|.100 V/750–1,000 mA, 30 min
• Wire electrode...10 V/30–40mA, 16 hr..|.100 V/380–500 mA, 60 min

Trans-Blot SD cell............N/A.....................|.Mini gels: 10–15V/5.5mA/cm2, 10–30min

......................................................................|.Large gel: 15–25V/3mA/cm2, 30–60min

These conditions work for a broad range, from large peptides to medium sized proteins and most large proteins. If you have very large proteins >200 kD in high percentage acrylamide gels, you may need to include a small percentage of SDS in the transfer buffer.

Note that several answers in this thread have incorrect information about SDS. It is essential during gel electrophoresis to maintain and prevent re-folding of proteins and allowing their charged residues to remain accessible to the electric field which promotes migration within the SDS-PAGE gels with a variety of buffers (e.g., Tris+glycine, Tris+tricine, MOPS, etc.).

During electrophoretic transfer to membranes (PVDF, nitrocellulose) SDS is NOT needed. In fact, it can block the absorption of proteins/peptides to the membranes, and you will loose some sample that doesn't stick well to the membrane into the buffer. Instead, in standard transfer buffer (Towbin) METHANOL is added to Tris+glycine. It helps remove the SDS from proteins as the leave the gel so they can stick better to the membrane. It may also help fix the proteins/peptides to the membrane.

However, in very large proteins in high concentration acrylamide SDS-PAGE gels, there can be some difficulty in them being transferred out of the gel and onto the membrane. In these limited cases, SDS can be added to transfer buffer to promote.

Of course, in PAGE and transfer of native proteins SDS cannot be present at any time.

A comment about temperature in tank systems... if running low voltage/current overnight, the system does NOT have to be cooled (e.g., run in cold room, refrigerator, or with cooling coils). If running at high voltage/current for minutes to hours, then the system does heat up and it has to be chilled. If it fits, it is helpful to put in a magnetic stir bar in the tank and place it on a stir plate during the run. That helps maintain uniform heat and uniform buffering, preventing variability. High amounts of MeOH in the buffer changes the resistance and increases the heat a lot during high intensity transfers to cooling is essential in these cases. (Things are somewhat different in rapid, semi-dry transfer systems. In those cases you must follow the manufacturers instructions EXACTLY or you'll ruin you gel, your transfer, and maybe the equipment as well.)

A final tip. I switched over from nitrocellulose to PVDF decades ago and never looked back. PDVF is superior for most applications and is not brittle like nitrocellulose that can rip unexpectedly. Just remember to pre-wet the PVDF in MeOH before placing in the transfer buffer of your choice.

In the original question, the user lost his mid-sized protein during the transfer. He had SDS present and was using nitrocellulose. After reading the above, one can understand how and why this happened.

(I apologize for the dots, this web reader deletes extra spaces and the periods are needed to align the columns for a table)

________________

See this archived PDF file for complete information:

https://web.archive.org/web/20180328165521/http://www.bio-rad.com:80/webroot/web/pdf/lsr/literature/Bulletin_2895.pdf

I usually use 250mA constant for 90min. the protein bands are transferred successfully