I'm trying to express and purify a small, 6 kDa protein in E. coli and was told to detect expression I needed to run a 20% PAGE Gel. I've never used anything that thick before, and most often use precast NuPAGE 4-12% gels, so I wasn't quite sure what to expect. When I ran the gel the first time, I ran it at 90V for 4 hours and stained with Gelcode Blue. Strangely, the dye seemed to be taken up by the gel better than the proteins (see attachment), and the bands weren't very distinct. So the next time I ran the gel including a 4% stacking gel, ran at 150V for 2 hours, and used a silver stain. Now I can see the bands great, but each well is frowning severely and I don't get any staining below 10 kDa.
Any thoughts as to what I might be doing wrong? The gel is not running hot, as the solutions are all cool to the touch and current is less than 90 mA, and the gels are fully polymerized. Sorry if this is an obvious issue, but I haven't been able to find anything to help me so far.
Thanks in advance for any advice!
hello! I would advise using tricine as the cathode buffer for the separation of such a small protein; increasing the amount of acrylamide over some point just doesn't give one a satisfying resolution... There is a recipe in the nature protocols for the tricine-sds page and if i remember correctly you can get all the details there..
Hi, have you already tried a less concentrated gel like 12-15% but just to run it for 1h-1h30 at 100-120V to be sure your protein didn't get too far? I've already worked with 15% gel for a bigger protein and get a good separation. I had a 4% stacking and runned the first 20 minutes at 100V and then get up to 120V for the end of the migration.
Good Luck
Hello!!. As Ana say, tricine-PAGE would be better for your small protein size (Schägger H, vonJagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1-100 kDa. Anal Biochem 1987;166:368-79). Also you could try a 10-20% gradient Tris-Gly gel. Good luck....
The Schagger-von Jagow protocol (with Tricine buffer), as described in Analytical Biochem 166 (1987) 368 (see also Nature Protocols 1 (2006) 16) is the preferred protocol for separation of small proteins in an SDS-gel.
When running cell lysates on an SDS-gel, the massive amount of phospholipids in the lysates often causes blurring of small proteins in an SDS-gel. Try to avoid accumulation of phospholids in your lysate,. eg.don't use soaps (SDS, Triton-X100) to prepare your extracts.
Good luck.
You should use tris-tricine SDS-PAGE as suggested. But according to the gels, it seems there is another problem than the gel concentration. Boil the samples properly (ca 10 min) and definitely use stacking gel. I analyzed such a small protein several times and I used14% gels. It worked perfectly. I always use constant voltage 200 V for whole run (ca 50 min). Since every protocol is different I can send you the one I used. Just dont know where to get your adress.
I think the problem lies in the high amperage you use, tried with 30 mA and a voltage of 150 V only
To prevent your protein exceeds the gel, use a front line with bromophenol blue, blue angry he therefore visible to the naked eye and allows you to stop the migration once the blue arrives at level at the end of your gel
You definitely should try a 15% gel, in my lab we work with proteins of 3kd and they get perfectly separated and visible in this % range... I think 20% is just too much. We use MOPS buffer to run the gel and boil the samples 5 mints. Oh, the stacking we use is 5%. Staining 5 minutes with Comassie works fine for us. Good luck!
It is better to run on 10-20% tricine gel. Novex Tricine gel, Invitrogen.
It is easy to resolve the small protein by running on Tricine gel. Novex tricine gel, Invitrogen.
Hi jason, yes, it is a major problem observed in separation of low mol.wt protein bands. I am sure neither high conc. SDS-page, nor Tris-glycine gels will be effective in obtaining efficient/high resolution of low mol.wt protein bands. We have just submitted a manuscript to analytical biochemistry i suggest you to read the paper as soon as it comes out.
Thank you, sorry for making you to wait.......
with regards
Rajagopal.k
Hi, as already mentioned by other people, 10-20% Tris-tricine (Invitrogen) give good results for small proteins. Also reducing your sample (DTT, Mercaptoethanol,..) will give you good results in case your protein might have tendency to form polymers which shows high molecular weight bands as in your gel.
In addition to other suggestions, we try lower voltage (30 -40V) over night for new protein experiments. After first time running, the proper voltage will be predictable.
I understand your problem of working with such high gel concentration. It is worth trying to run the gel with 1:1 and 1:2 diluted buffer in the tank. Low electrical conductivity, low voltage and longer duration of electrophoresis (for 6-8 hrs) helps you in keeping the temperature of the gel low. If the temperature shoots up, you get such distorted bands.
I also suggest that before staining,treat the gel in 1:1 mixture of 50%TCA and Isopropanol overnight on an orbital shaker. Such pre-treatment will fix very small peptides efficiently. With a brief wash in distilled water, transfer the gel to stain.
With the pre-treatment, the gel will shrink; but don't worry. When you put it in stain, the gel will come to normal size.
Please refer to my paper published in Biochemical Genetics 27:507-520 (1989)
Hi! Jason,
A 14-15% gel should take care of your 6kDa protein. Also make your lysate in sample buffer having bromophenol blue to track the dye front while running the SDS-PAGE. Important thing is try running the PAGE immediately after making the lysates without freezing the protein lysates. If you are freezing also freeze in aliquots and don't do repeated freeze, thawing. Stacking of 5% is fine. Run the gel at constant current of 20mA and see. Then try the coomassie staining of ur gel. It should work i guess.
Good Luck,
Madhavi
Wow, thanks for all the responses! This place is awesome!
@Ana: I found the nature protocol for tricine gels, and that sounds like a good option. Thanks!
@Louis: I have indeed tried a lesser % gel, but only a precast 4-12% gel. I get good bands and staining but all the proteins under 10 kDa seem to bunch up and I don't get any separation. See attachement.
@Hector: Cool, another vote for tricine, sounds like that may be my next step.
@Walter: Great tip on the soaps, my lysis buffer actually has both SDS AND Triton... Could be a big part of my issue!
@Michal: Boiling: check. I'll give the tricine a try and if I still have issues I'll check in with you for that protocol. Thanks!
@Yahia: Amperage is a good consideration, I'll be sure to pay attention to that when I try the tricine.
@Dewald: An interesting idea. I'm using laemmli buffer with bromophenol blue. Does anyone know at about what comparable protein size it runs?
@Marcin: 15% with 5% stack sounds a lot easier than tricine, which I don't have. Maybe I'll start with that... decisions, decisions...
@Wim: Thanks, I am using BME, so that shouldn't be a problem.
@Saraf: Interesting idea, I didn't think that maybe the small proteins are diffusing away. A fixing step could help prevent this, I shall consider it thanks!
@Madhavi: Good tips, thanks!
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You guys rock, seriously! Some great ideas here. I think my plan is:
1) 15% with 5% stack on constant current. No soaps in the lysis buffer. Boil and reduce samples before loading. Move immediately to fixing and staining so no diffusion of small proteins can occur.
2) If that doesn't work, try the Tricine gel.
Thanks again everyone, I'll update you when I have more info!
for small proteins I use Nupage gel in Mes running buffer (invitrogen) have a look
http://www.lifetechnologies.com/order/catalog/product/NP0002
http://www.lifetechnologies.com/content/dam/LifeTech/migration/en/filelibrary/pdf.par.99253.file.dat/o-063575-nupage-fin.pdf
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