Few things about blue native page I couldn't find at all. First, why do we have separate cathode and anode buffers (in traditional SDS PAGE tris-glycine is the only buffer) in BN PAGE?. Moreover, there's plenty of coomassie g250 in the loading buffer, then why do we need 0.02% coomassie in the cathode running buffer? Can the gel be run just with tris glycine buffer on traditional tris-HCl gel (stacking gel pH 6.8, resolving gel pH 8.8, running buffer pH 8.3) if we add coomassie g 250 in the loading buffer (the whole system in this case will be devoid of any SDS).
Another question comes to me is about the molecular weight ladders. If we coat the normal standards (commercially available, meant to be run on SDS PAGE) with coomassie G250, they should run across the gel. In principle, these standards should give approximate estimates of molecular weights in BN PAGE. Kindly correct my thoughts on this issue.
Hi ! Here something about SDS PAGE and what happens there http://mullinslab.ucsf.edu/Protocols%20HTML/SDS_PAGE_protocol.htm
In BN-PAGE you want to keep your proteins in a native state so i wouldn't recommend a change in pH as you might denature them.. The Coomassie in the buffer keeps the charge in the proteins (i guess so) so that they even run (no charge = no mobility) and you have to have a native protein marker as SDS markers contain denatured proteins which run completely different than native ones as they have a different shape and thus mobility on the gel.. For further reading http://stke.sciencemag.org/content/2006/345/pl4.abstract and http://www.nature.com/nprot/journal/v1/n1/full/nprot.2006.62.html hope this t helps
Thank you Norbert. It's a coincidence that I have already thoroughly read these 3 links (and many other resources on this topic!). I also checked the original paper by the inventor of this method (Scha¨gger 1991). My first question still remains unanswered. Nowhere the authors have bothered to explain the need of two buffers (cathode buffer and anode buffer). Their compositions are different. The impact they have on electrophoresis is also unknown.
I agree with you on the importance of keeping pH around 7.4 during electrophoresis. This is the reason, they recommend bis-tris or imidazole-HCl buffer for gel and the running buffer instead of traditional tris-containing buffers. The inclusion of tricine instead of glycine as a trailing ion is for better separation of low molecular weight proteins.
You are right, the SDS PAGE markers contain proteins that are already denatured. These ladders/markers have 2% SDS in them. Therefore they have uniform charge and will migrate as per their molecular weight. The coomassie G250 concentration in loading buffer also equals to 2 % or even more (protocols recommend the addition of 5% coomassie G250 that will give dye-detergents ratio of 1/8). As a result proteins coated with coomassie G250 will also migrate according to their molecular weight. In addition their migration will also be affected by their molecular shape. Therefore, migration of the SDS PAGE ladder and the coomassie G250 coated proteins will be approximately comparable (not exactly because of differences in molecular shape-ladder proteins are rod shaped while test proteins are in their native conformation).
On the other hand if we use standard native page markers, they will have their own coomassie G250 binding capacity. Their migration cannot be compared with migration of our protein of interest. I understand, that use of SDS PAGE markers in blue native page is strongly condemned throughout the scientific forums but no one is carefully scrutinizing the logic behind these widely accepted notions.
Hello! I would recommend you not to bother about Blue Native PAGE. This method was invented for separation of protein complexes and actually works, but for only very tight protein complexes. If you need just the native PAGE - use old classical Weber&Osborn PAGE - this is the method which Laemmli modified by adding SDS. It's funny but some people anecdotically call this method - Laemmli's PAGE without SDS.
Sorry for confusion. I kept in mind Ornstein & Davis disk electrophoresis (1964) when was talking about native gel electrophoresis. Weber&Osborn (1969) were the first who introduce SDS in electrophoretic separation.
Thank you Dr. Naryzhny, isoelectric point of my protein in un-phosphorylated condition in 8.95. It won't run across the gel if I exlude SDS from the PAGE. I once tried to run the gel without SDS (Native gel as you mentioned). All I got is smear. It was 4% stacking (pH 6.8) 6% resolving (pH 8.8) gel. Running buffer was tris-glycine pH 8.3. Please find the attached results. The molecular weight of protein is 43 kDa (its connexin 43 protein). It forms hexamer that I want to detect.
Hello, Tushar! You are right, your native PAGE is not very good. Is it a run of the pure protein? It looks like it's very sticky, so this is the problem, not only the charge. But as I guess you would like to keep this protein in native condition and find how the phosphorylation is involved here. Anyways, how your SDS-PAGE of this protein looks like?
Please find the attached image of Western blot after SDS PAGE (3rd slide in the presentation). My aim is to see the band that represents hexamer of the protein. Hexamer is the physiologically active form of Cx43. Therefore, I performed native PAGE with 6% resolving gel. In all cases, I used mouse hippocampal lysates (clear supernatants after lysis and centrifugation). No pure protein used.
Hello Tushar! Did you try crosslinking? Formaldehide for instance. Sometimes it works, if you are lucky.
Maybe this thread is over, but anyway - if your protein won't run because of its high pI, can you prepare a running buffer with a higher pH that will allow the protein to run?
First to answer the thread starter question. The loading dye contains Coomassie G250 to coat the proteins. If your protein isoelectic point happens to be 4 (for example, positively charged), and once it reaches the gel matrix correct molecular weight position, the protein will stay in that place but the CBG will still continue to move downwards. The dye will then start to detach itself from your protein. Your protein now is positively charge and will move back up the gel, That is why the cathode buffer requires a continuous supply of negative charges to keep positively charged protein locked in the gel matrix.
Blue Native PAGE uses Coomassie G-250 (NOT R-250) as a charge shift to coat proteins with negative charges. This anionic dye binds strongly to proteins, but do not denature them as with SDS.
You will need to identify your protein isoelectric point before attempting to run clear native PAGE (no G250 or SDS). If your protein isoelectric point is about 9, you cannot use Laemmli system with pH 8.8. The proteins will hardly move since there is no charge. Try using acidic PAGE or neutral PAGE for this purpose.
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