Hi

I usually transfer 3.5 kDa protein separated on 16% Tris-Tricine gel. I recommend not to add SDS in transfer buffer. Itransfer them at 70V for 2 hours and get good bands. see the attached fig

I am studying the effect of some drugs on the activation of protein of interest,

the proprotein has a molecular weight about 26 and once its cleaved it gives a product with 10 to 20 KDa. in my work I can not do tagging.

Have you tried blotting onto 0,20 µm PVDF? Its retention is better. Perhaps you should also reduce transfer time. A good try is always to put two membranes, one bellow the other in the transfer cassette, so if the proteins get through the first membrane, you could always get them on the second.

I don't think transfer will be an issue with 0.45uM nitrocellulose membrane. All proteins are hindered from binding to membranes by SDS but small proteins more so than large proteins. If your protein of interest is small as you mentioned, consider removing SDS from the transfer buffer and keep the methanol concentration at 20%. Also if you are using semi-dry transfer then you can try wet transfer (12hr ,constant current of 120mA) which is more efficient. Also try higher percentage of gel like 15% to make sure protein didn't get out of gel. You can also try some other protein antibody of smaller mol. wt. to confirm that your antibody is fine for detection as well as lysate which u r loading in enough in concentration. As many proteins have very less expression then you may need more amount of lysate to detect their expression. Finally you can check the literature to search than if someone else have done those western then what condition they have used or you can communicate to them if not mentioned in details.

Hope it will help..........

Gradient SDS-PAGE (4-20%) might help.

My simple solution is similar to some of the above comments. I use PVDF, which is actually 0.2 um pore, or 0.2 um nitrocellulose membrane for the transfer. I will recommend you to use 200-220 mA (~25-40V depends on what kind of transfer system) transfer time for 60 min on ice. Judging from so many small proteins I will recommend AnyKD gel or 4-20% gradient gel that can give you a better separation. If the proteis are lipophylic than PVDF may be the better choice. Hope it will work for you.

I guess we also need to consider what loading control to use. If high MW protein is used then 30 min may not be enough. But, 100V may provide 250-300 mA current so 30 min may work for low MW proteins.

As some have said above. PVDF is far superior compared to nitrocellulose. With your very long transfer duration (would definitely decrease it) the small proteins go right through your membrane. Have you got any low molecular weight bands in your marker that you can use as reference to see if that really is the case of proteins going through the membrane or whether you ran them off the gel? I have no problem seeing my marker bands as low as 2kDa on my gels.

maybe low temperature 4C 22V transfer over night would help.

Use appropriate gel 8-12% , .2 micron nitrocellulose wud be most convinient for detectiion

I see the band of the protein marker at 10 KDa on the nitrocellulose membrane after the transfer for 90 min at 90 V. I tried the overnight transfer too at 20 V and 4C but it did not work, I was using 15% gel at that time. my looding control is GAPDH (37 KDa) so I will try 30 min transfer and try to detect the cleaved caspases. I hope it will work. I am using a wet transfer with 20% methanol without SDS

Maybe your caspases are not being cleaved, so that´s why you don´t see those sizes in your western... Or you´re not loading enough protein extract...

I load 20 µg/well but in literature this drug showed cleavage of caspase 9 and 3

Hi

I usually transfer 3.5 kDa protein separated on 16% Tris-Tricine gel. I recommend not to add SDS in transfer buffer. Itransfer them at 70V for 2 hours and get good bands. see the attached fig

Hi,

I agree with Ahmad's method. Your best bet would be to use a 16% Tris-Tricine gel and a Tris-Tricine-SDS running buffer. Thereafter, you can transfer onto a 0.2 microM PVDF/Nitrocellulose membrane using a buffer without SDS at 100V for 1hr for precast gels or 1hr 45 min for hand-poured gels. This is our standard protocol for doing Westerns on peptides and it has always worked without any bleed-through issues.

You can please follow the links below to obtain valuable informations elaborately:-

1. http://web.mnstate.edu/provost/ProteinBlottingBook.pdf

2. https://promo.gelifesciences.com/gl/WESTERN-BLOTTING/misc/28999897AB-WB-HANDBOOK.pdf

3. http://www.bio-rad.com/LifeScience/pdf/Bulletin_2895.pdf

Can any of you comment on the effect of using different sample buffer recipes, when it comes to small protein fragments?

Did anybody detect 2 kDa peptide protein using wide range gel?

Which method of transfer you use: dry or semi-dry, and which is better for small proteins?

Novex™ 4-20% Tris-Glycine or 16% Tris-Tricine gel

0.20µM nitrocellulose rather than 0.45µM

100V for 30 minutes in ice-water

Mr Waqar Ahmad, I am really interested in your method of western blotting. I am having difficulties at detecting small molecular weight proteins. If you send us your western method with all small details I will be grateful.

With regards

Meryem

I faced with the same problem I also want to detect small weight proteins including cleaved caspases. I couldn't get them anyhow. For my other proteins bigger than 25kDa they all worked well after 90V transfer for 1hr in 0,45um PDVF membrane. So I want to know finally have you solved your problem. AND HOW? Which voltage and currency are you using and how long for the transfer? Is there any special component of the transfer buffer?

Thank you very much!

Hi... Can anyone tell me the conditions(Transfer time? Voltage?)for wet-transfer to get cleaved Caspase-1. I have tried many times but not getting.

Dear Waqar , will your method work with 0.45um nitrocellulose membrane for a 6 kDa protein

I do usually run 12% Tris-Glycine buffer 70V /2h. Transfer with 20% methanol (without SDS)

100V/ 1 h using 0.2u nitrocellulos.

it works with me since I’m interested in 17kDa protein

I recommend to check sample preparation

regards

For