I did a good paper visualising 2 kDa peptides (Tris-Tricine and 16 % acrylamide) and then blotting them on PVDF fro anitibody staining. All the suggestions are OK! however, from your question, I wonder if you are searching for methods to separe complex peptide fractions. In this case, probably an isoelectrofocusing gel electophoresis could be of help. Especially, you could inverse the polarity (just invert the red and black jack) and let the proteins run up to the bottom positive pole, using red coloured cytochrome C as marker of basic pI (10). This method resolves complex pattern of proteins independently from their size.

Hello,

A 12 or 15% polyacrylamide separation gel should give you good results.

Good luck

Dear Plundrich,

Normal 15-18% SDS-PAGE gels can resolve your proteins (

Tricine gels or bicine gel

agreed....using by 15% polyacrylamide will help you to obtain a good results. Good luck..

20% acrylamide standard glycine gels or 10-15% acrylamide tricine. There are quite a few common tricine recipes out there.

use Bio-Rad Criterion precast 15% Tris-HCl gel, run it with Tris-Glycin SDS buffer at 170V for ~60 min. You shold see your protein in the bottom half of your gel.

Good luck

one more thing if your protein is smaller than 11 kDa it would be better if you will use 18% Criterion gel

15% polyacrylamide separation gel should give you a good result and i suggest the same think as Mr Upendar Golla to avoid high salt concentration in protein samples which creates real problem during migration. good luck

15% Tricine-SDS-PAGE is enough for me. Pls. try & good luck

I agree using Tricine gels, with almost 15% separation gel. In fact, the proteins in the lower molecular weight range tend to have an higher mobility than glycine. Tricine used as trailing ion, migrates faster than glycine so it gives a better resolution.

I m highly suggesting for you to use Tricine SDS-PAGE following nature protocol by Schägger 2006. In case still not having desired resolution, u can add the spacer gel between stacking and separation gels.

Oleksandr, and I guess to everyone else too, I have been using Bio-Rad Criterion Precast Tris-HCl gels (10-20% polyacrylamide) and Tris/Glycine/SDS running buffer, 200 V, 55 min....if this info helps anyone. I have the proteins/I think at least most of them on the gel, but I think or hope I can get a better resolution or let's say a particular look at especially the

Tricine gel resolves the smaller protein size with much more precision and better resolution than the glycine system.

Tris-tricine indeed is good for small proteins ..but for a better resolved separation in the gel you should work on sample preparation...I understood that you have proteins not a particular isolated one.

Novex tecnology of course (invitrogen) tris acetate gels ;-)

Invitrogen gel 12% with MES/SDS buffer, Transfer with 20% methanol on 0.2 microns nitrocellulose paying attention not to overtransfer

I see that other scientists suggested the same method with tricine, we also used (carefully) a gel at 6%, anyway go to "LifeTein, The Peptide Synthesis Company"

From my experience, blotting of small proteins is more of a problem than resolving them on a gel. With DAP10, we only succeeded using wet transfer over-night onto PVDF membranes. We had an N-terminal epitope tag, so we new the cells were positive by FACS, but blotting it was the challenge.

Hi Nathalie,

I did have good experience with homemade 14% separating gels (total length ∼12.5 cm) overlaid with 4% stacking gels prepared from a stock solution of acrylamide:bisacrylamide = 40:1.7. Electrophoresis was carried out at room temperature at 180 V for ∼3.5 h, and continued at 260 V until the bromphenol blue reached the bottom of the separating gel, and further continued for an additional 1.5 h. It is long run but I've got good separation among 18.7, 17.2, 14.8 and 14.3kDa proteins if you are interested I can send you the protocol.

I would also suggest the Shagger tris-tricine system. Specifically our homemade gels were made of a separating gel (16.5% T, 6% C) overlaid by a 10% T, 6% C spacer gel and a 4% T, 6% C stacking gel. In our hands, this 16.5% tricine-PAGE worked just fine with proteins/peptides down to 1 kDa.

To avoid peptide leakage from the slab gel during staining (we lost peptides 1-4 kDa), we fix the gels with 5% glutaraldhehyde, before staining with CBB-G in 10% acetic acid. The run were at 30 V until the samples left the wells and then at 110 V for about 4 hours.

Hope this helps.

Hi Nathalie. Great to hear that you're achieving some success. With the percentage range of precast gels you are using, I believe you will have no problem in resolving a protein of that size. As previous scientists have suggested, 12-15% should do. May I suggest some tips from our lab that you can use to improve your resolution. The first thing is to remove any bubbles that may have appeared, when you added the running buffer to the outside of gel tray, at the bottom of your clamping frame. They can be removed using a syringe and needle. These bubbles interfere with the electrophoresis. The second thing is, as I understand, precast gels also have the stacking gel component, so what you need to do is run your samples at 60V for about 60 min or just until your sample reaches the interface between the stacking and resolving gel. Once your sample has reached this point, increase the voltage to 120V and electrophorese until the end which usually takes about 2-3 hours. I hope that these tips also assist you in achieving your desired result.

Best of Luck!

a simple trick , is to double the amount of tris in the separating and stacking gel...I don't not why, but this way you increase the range of mw you can visualize by PAGE SDS...in a 12% gel, you have a 6kda protein!

Thank you all so much for your suggestions! Appreciated. I will try to use some of your tricks and see if it's working for me.

I am working with a 4 kDa protein in 15% Bis-Tris-SDS gel or Invitrogen gel 4-12% with MES/SDS buffer. I transfer with 20% methanol on 0.22 or 0.45 microns PVDF membrane.

I have tried some 16% Tricine gels from Novex we had readily available and as far as I can tell for SDS-PAGE it seems to be working out. Will try Western next. Thanks so much for all your valuable input!

Use a 15% Tris-Tricine gel but don't let it run till the end. Stop it before it leaks

Yep, this gel system worked great for my SDS-PAGE and Western. I can recommend it.

Dear Nathalie

You can use 20% SDS-PAGE according to the following article:

Molecular & Cellular Proteomics 2:1096–1103, 2003

Please pay attention to the following image:

I did a good paper visualising 2 kDa peptides (Tris-Tricine and 16 % acrylamide) and then blotting them on PVDF fro anitibody staining. All the suggestions are OK! however, from your question, I wonder if you are searching for methods to separe complex peptide fractions. In this case, probably an isoelectrofocusing gel electophoresis could be of help. Especially, you could inverse the polarity (just invert the red and black jack) and let the proteins run up to the bottom positive pole, using red coloured cytochrome C as marker of basic pI (10). This method resolves complex pattern of proteins independently from their size.

Hi Nathalie, Wish your protein resolution is proper now! Simply, increasing thee percentage of separating gel, say above 15% should solve the problem and as you have mentioned, you are using 10-20% SDS-PAGE gels. That's enough to give a good resolution. Good Luck.

The best:

http://www.nature.com/nprot/journal/v1/n1/full/nprot.2006.4.html

Simply, you do a SDS-PAGE with 15 % Acrylamide. Good Luck.

12-14% U CAN USE the thing is that u have to run till the end. i have used 12% for 17 kda protein. it was resolved nicely.

Dear Nathalie,

I have used the Tris-Tricine system and it works exceedingly well. Because I've was concerned about my protein coming off the gel...really easy in a constant gel %, since I always made my own gels, to include mini-gels, I was preparing 5-15% gradient gels and able to resolve very small macrocyclic ring compounds secreted by Pseudomonas syringae along with very low molecular weight standards. As I remember, I also tried running a quasi-discontinuous gel where there was a gradient on top of a segment of the gel which was of higher acrylamide %. Entry into this lower region...need to add the gradient right on top of this constant % so you don't get smearing at the junction...there was better separation of the closely related molecular weight compounds. I tried this with a student working with me and it worked. For the most part, however, the gradient gels were best for our application.

Good fortune.

David

i used1X MES buffer and i could detected so easy the Protein band below 15kDa.

the components of MES buffer : 50mM MES, 50mMTris-Base, 1mM EDTA, 0.1%SDS. pH7.3

You can go for Tricine SDS PAGE rather than Glycine SDS PAGE. Please find attached the article from Nature for your kind reference. Hope this will help you solve your problem... Good luck for your research.....Best regards

hi,

u can make SDS-PAGE separating gel mix for 12% and stacking gel mix for 5%

if u need complete protocol send me ur mail id

gud luk

A 15% medi gel works fine for my ~12 kda protein

Hi Nathali,

You need at least a 12 % polyacrylamide gel. 15% works well also. If you are currently using Tris Glycine buffer, changing over to Bis-Tris Gel with MOPS or MES buffer will give you another variable to consider.

I use 15% SDS-PAGE, for transferring to membrane PVDF (0.2micron) membrane is used, which should be methanol (without any dilution) soaked ( for 30 sec) and immediately equilibrated with transfer buffer. It works fine for me to detect 13.5 KDa protein.

I used 16% tri glycine gel to get good resolution from 3 to 20 kDa protein peptide bands.

I still can detect 14kDa Angiogenin1 in 4-12% gradient Bis-Tris gel using MOPS-SDS buffer, but if one decides go below 14kDa, I would recommend MES buffer and not to increase the percentage of the gel, as it would be harder for proteins to leave a gel.

Hello Nathalie,

I am sure you can run 12-13% SDS-PAGE gel, and if you are trying to look for particular protein of interest below 15 kDa, transfer the gel on PVDF membrane for not more than 25-30 min (im sure you know that before setting up the transfer, PVDF membrane is to be prewetted in methanol and equilibrated in transfer buffer for 10 minutes). Transfer at a constant current calculated according to the dimensions of your gel. Later, you can probe and see whether your protein is present or not. Also, I have experienced that proteins run as soon as sample preparations were made gave better results than the once which are frozen and run later.

Good luck with your LMW proteins

Madhavi

You can use 15% gel

12% to 15% gel strength can be use for high molecular weight protein

I am agree with Dr. Pradip.

I agree with above, try 12-15% (15% recommended) and when you transfer, make sure not to over-heat the gel or membrane