@ Tushar Deshpande. I saw the diagrams you provided with the question. Probably the problem is with the separating gel. Ensure that the polymerization( in both stacking and separating gels) is complete. Secondly, one may encounter similar problem if the salt concentration is too high in loading prep. But!  the ladder is also smudged... so i guess u need to check with the polymerization in the gels.

hope this helps!

Thanks Jayanth. I have used commercially available gels. These are native  gradient (4-16%) gels. Polymerization problems are rare with such gels. Salt concentration is something worth to be considered here. Product manual recommends maximum 50 mM NaCl in protein lysis buffer. I have kept it 40 mM. It gets further diluted with loading buffer which does not have NaCl. Still the streaking is present.

I am not sure as to why you are getting streaking - perhaps overloading or heterogeneous post-translational glycosylation? It certainly looks like you have a lot of protein loaded there.

As to the gel not running to completion - often at 250 V, the current drops too low for the power pack to deliver. In that case, you can increase the voltage to 300 V or even a bit higher to get the gel to run to the bottom. 

Hi,

The problem is that you probably did not use the proper buffer composition for BN PAGE (if it is really what you want to do).

You need to have 6-aminocaproic acid in your gel and sample buffer (500 mM in the gel) to prevent protein aggregation during electrophoresis. (see: Schägger, H., and von Jagow, G. (1991). Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal. Biochem. 199, 223–231.).

BN-PAGE kit are commercially available and will help you to start.

For western blotting after BN-PAGE, you will need to replace the blue running buffer by a clear one when migration reaches 2/3 rd of the gel. An other option is to un-stain the membrane after the protein transfer and then to immunoblot. It will remove a lot of background and improve signal as some antibodies does not "like" the presence of Coomassie.

The fact that the gel does not run completely is not an issue. As the protein complexes are in there native state they will stop migrate when the acrylamid density exceed a certain level. In other word, if you leave your gel running overnight the complexes will not move any further in the gel.

I hope it will help you.

Factors affecting native gel quality (from experience): Running condition temperature, having a stacking gel, current used for stacking vs separating gels, age of the gel, protein quality loaded, sample salt components, nature of protein (glycoprotein, lipoproteins, etc), running buffer concentration, and others. The image you had it 

Hi Tushar! I think all the answers are helpful, I would just add that I believe the acrylamide percentage you are using is too wide, I use 4-12% for separating a 1,600, -800-400 KDa, complexes. I don´t know if there are other percentages available but I would suggest that maybe another one with a smaller gradient could give you a better resolution. Also I believe Patrick is right about loading buffer, I use 50 mM epsilon amino-caproic acid and 50 mM Bis-Tris pH 7.0. 

Thank you all.

1) @ Roger Bradley Dodd- Overloading could be an issue although we do not see an improvement in streaking when protein amount is halved (from 10 to 5 ul). I will try further reduction in loading amount. Voltage limit of biorad system is 600 VDC and 15 watts. I never went beyond 250 V, will try higher voltages.

2) @ Patrick Paumard Yes, I definitely want to perform BN PAGE. Regarding  amino caproic acid (also known as 6-aminohexanoic acid), indeed original article recommended it for better solubilization. However, same authors, in their article in Nature protocols 15 years latter write that, "6-Aminohexanoic acid is not essential for BN-PAGE, but it is an efficient and inexpensive serine protease inhibitor" (please find the attached article, sentence highlighted in green). Since I am adding protease and phophatase inhibitor cocktails to lysis buffer, additional protease inhibitor is redundant. However, if amino caproic acid is indeed acting as a solubilizer or aggregation inhibitor, then I can try to see if its addition prevents streaking.

I am following the gel running protocol exactly as you mentioned.

I need not to worry about abrupt stoppage of gel run if it is 'normal''.

3) @ Arshad Jawed - the product seems to be suitable only for 2D gels.

4) @ Alexa Villavicencio Queijeiro, I am not sure if changing gradient will help to resolve streaking problem. It may help to get better resolution when protein is perfectly soluble throughout the run and when there is no streaking at all.

Hi Tushar,

As per your protocol I too think you need to add 6-aminocaproic acid (6-ACA). Incase you want to inhibit serine protease add a protease inhibitor cocktail in your extraction buffer. Moreover there is no need to add detergent in your loading buffer just 6-ACA + Bistris + Nacl is enough. I don't use NaCl but I have come across protocols using NaCl hope this doesn't matter. May I know do you add glycerol to your sample while loading ? As you mentioned the glycerol in your excel sheet you provided, I'm not clear of it. In case if you are adding glycerol to your sample I think that will be a reason for such a result. Since I've also coming across such a problem due to the triton X-100 in my sample more than that of  expected CMC to present. Anyway I would suggest my publication to refer to the protocols, even in that case you have doubts you are welcome.

Hippocampal receptor complexes paralleling LTP reinforcement in the spatial memory holeboard test in the rat

Best wishes

Thanks Saraswathi. Yes I am adding glycerol to a final concentration of apporx. 10%. There are phosphatase and protease inhibitors in the extraction buffer. There's no triton x anywhere in the protocol. I will give a try with your protocol, thanks again!

Tushar was the problem of streaking solved?

If it did then what is the final protocol used.  It would be great help if u share that

I use the same system for membrane proteins and I have encountered all of the same issues. Here are a few factors that improve these issues:

1. I keep my anode and light blue cathode buffers at 4 degrees, and I prechill my dark blue cathode buffer for ~30 mins prior to running the gel (concentrated coomassie eventually starts to aggregate after a while at 4 degrees, so keep the dark blue buffer at RT when storing).

2. I run my natives in the cold room (4C). This makes a huge difference in the resolution of the bands and the streaking in the lane. 

3. The gel stopping before the end is common, I think it's some kind of incompatibility between the novex gel box and the power supply? I just cut the extra part off the bottom since there's no way around it when running just one gel. If you run 2 gels at the same time, however, you will be able to run them till the end if you increase the voltage to 300mV.

4. This novex system is designed to work with only certain detergents, otherwise things are prone to streaks and aggregation. I use LMNG (similar to DDM) which works well. Novex recommends DDM or digitonin (check the site for more info).

5. The Native Mark ladder really stinks. I don't know any way around this, other than complain to the company because they should offer a better product. I just run another more stable membrane protein that expressed well in HEK cells and that I know runs at the same MW as mine as my 'standard'.

6. Lastly, I recommend (if you don't already do this) filling all of the wells a couple times with dark blue cathode buffer to 'rinse' out any glycerol and whatnot left around from storage buffer and to make sure salt levels are consistent from well to well. This also makes the wells visible so it's easier to load your sample. Then I add the dark blue cathode buffer to the inside reservoir after loading my samples.

Tushar Deshpande did you get this fixed? I also have a problem with my proteins not leaving the wells. I followed exactly the protocol from Thermo and used their sample prep kit prior to loading my samples. How did you lyse your cells? My samples are from Hek293T. I isolated my proteins using RIPA buffer, which was not ideal for NATIVE PAGE due to the detergents present but it did not seem to denature my proteins because they're stuck on the wells so I assumed they're large complexes.I have detected my protein of interest on SDS PAGE but I wanted to see it on native state to know if they come as dimers, trimers, etc.

beulah mae ann villalobos Solivio Can you try your protein prep with something other than RIPA? I've seen so many various small problems with preps using RIPA. Haven't seen it used with native PAGE yet but I used LMNG (very gentle but effective, great for sensitive membrane proteins) and DDM with blue native PAGE and had great results.