During electrophoresis, the temperature of the apparatus reached above 60 C. Most of the protenicious enzymes are denatured more than 50 C. Find thermal stability of your protein and run the electrophoresis with different concentrations by maintaining the temparature below the tolerant level. Surely you can get bands

Many thanks for your suggestion! I will try according to yours.

Does the bromophenol blue in the sample buffer run into the gel?

You should see protein staining in the sample wells, even if your proteins are not running into the gel. If you are not seeing any protein anywhere then you are probably using the wrong running buffer or gel%.

I would agree with Steingrimur, double check the buffer (and strength). Normally we run our native sample with a µl of coomasie blue(note sure,may be 1%) to facilitate the migration of protein by adding charge, in addition load a sample with denaturing agent(DTT or 2-ME) after heating for 5min at 95degrees. This will have to run as normal SDS gel.

We also run our native gels at cold room!

Good luck.

The protein bands was remained in the well (only for the marker). I used the bromophenol and make sure that it got into the SDS-PAGE.

Instead of larger one, try with miny slab gel apparatus with various voltages

@Van Dung Pham. Does the bromophenol blue run as a sharp band?

If it does then the gel% could be too high. If it doesn't then there could be something wrong with your buffer preparation. A common mistake is to adjust the running buffer pH with HCl.

I am so confused, I attached the gel picture here. Anybody can see clearly and help me. plz

I've seen worse.

Can you share the composition of your sample buffer, running buffer and how much salt is in your samples? Can you also verify that there is no SDS in any of your solutions?

I used :

1. 2x Native PAGE (30 ml) ( Sample Buffer)

62.5 mM Tris-HCl, pH 6.8, 40% glycerol,

0.01% bromophenol blue

0.5 M Tris-HCl, pH 6.8 3.75 ml

50% Glycerol 24.0 ml

1.0% Bromophenol blue 0.3 ml

diH2O to 30 ml.

2. 10x Native PAGE (1 L) ( Runing Buffer)

250 mM Tris, 1.92 M glycine, pH 8.3

Tris base 30.30 g

Glycine 144.10 g

diH2O to 1 L

Do not adjust the pH (~pH 8.3).

All chemicals were ordered from Biorad company and I make sure that no SDS in gel preparation.

Looks OK to me, except the high glycerol.

I gather that the buffer your sample is in does not have high salt either?

Well, if all else fails, toss out all solutions and make up a fresh batch.

First of all, if you are doing NATIVE PAGE then are you also doing the activity staining to cross check the presence of your protein. Run two gels simultaneously after reducing the concentration of your gel, proceed ahead with CBB staining as well as Silver staining. Also prepare the zymogram to detect the exact location of desirable protein. Proceed ahead with the freshly prepared chemicals and reagents. Also change the loading dye-prepare it fresh. Check the power supply unit too. Faint lines are observed with the ladder in CBB staining so it suggests:the conc. of protein loaded is less, increase the conc. or if you have less sample then go for Silver staining. Secondly the migration has not occured as desirably which might be suggestive of improper supply of current or a very high concentration of gel. Check the stacking gel concentration, because atleast the proteins should have migrated this region.

Check the sequence of your protein. In native PAGE, your protein has to have the necessary charge to move into the gel - if the pI of your protein is higher than the pH in your gel, or if the net charge is 0 (zero), your protein will move in the wrong direction, or not at all. And if you don't see staining with CBB, you can always continue and do a silver staining on the same gel, assuming you didn't put fingerprints all over the gel ;-)

Hi all. i'm also trying native PAGE for the first time and have a problem. I'm trying to see binding of a protein (approx 90KDa) to a small DNA fragment (30bp double stranded)...i.e. EMSA. I added ADP (100mM), NaCl (150mM) and MgCl2 (10mM) to my running buffer as i heard that you often need to include the co-factors for the binding reaction otherwise the binding is lost upon lane loading (basic running buffer is 25mM Tris, 190mM glycine). However when i run the gel i don't get migration of my tracking dye as a band. in fact it seems to dilute. also the current is very high relative to voltage (only 60 volts gives a current of over 110 mAmps). I assume this has something to do with the balance of the ions in the solution...can anyone give any advice? thanks a bunch!

correction...100 microM ADP

Asbit depends on the pI of your protein, only proteins with a pI acid can be seen ib this gels....in fact you can never see the ladder...So probably your protein migrates in the contrary direction

hi both. thanks for your answers. the pI of the protein is 5.49 and the overall charge is -14.8 so in theory it should migrate into the gel especially if it's bound to -ve charged DNA. i'm now getting a basic emsa result with a shift in the nucleic acid with reduced salt (only 50mM NaCl). However the protein/DNA complex is still barely getting into the gel. in fact the free DNA doesn't move that far...like 5-10% of gel length

I'm wondering if it's because i'm having to use a low voltage due to having so many small ions and thus current -> giving a weaker electric field and poor migration. I'm considering reducing the ion concentration even further. does that sound reasonable?

also do i even need glycine? i thought that was just required for the stacking effect and i have no stacking gel, just a single phase 5% gel.

thanks again

When I saw first native GE, it was in a horizontal instrument, and used agarose. Strange. Sampled in the middle to get possibility for both direction. Now in our lab others use vertical PAGE... If free 30 bp DNA can not run into the gel's end, start over from basic buffers and solutions.

Dear Van, if you still don't have a result with NATIVE-PAGE why you don't try alternative technique i experimented the MetaPhor Agarose is a high resolution agarose that challenges polyacrylamide ,i find it good and i finally get result .

http://www4.ncsu.edu/~rgfranks/research/protocols/Agarose%20gels/MetaPhor(18108).pdf

Good luck

i have been working on Native PAGE of beta-glucosidase for last 3 months but not getting bands on gel i have tried different protocols with different detection techniques but no results at all. what should i do? please give me some suggestions.

Hi Van, how the native western blot turned out then?

your question is very useful, hope you have worked that out.

J