Pre-cast Native-PAGE Novex Gels From Invitrogen

Native PAGE (PolyAcrylamide Gel Electrophoresis) gels are essential in the analysis of proteins in their native conformation. Native PAGE is an electrophoretic separation of proteins based completely on native size (monomer, dimer, etc) and finds its application in the field of proteomics. In addition to resolving any protein in its native conformation (non-denaturing conditions), these gels are also important for analysis of membrane protein complexes in their native conformations. Also, native PAGE finds its application in 2D analysis for resolving complexes, estimating the molecular weights of proteins and complexes, performing in-gel or solution activity assays.

The native PAGE gel system from Invitrogen is commercially known as the NativePAGE™ Novex® System. Options exist for gradient gels that are 3-12% gel or 4-16%; are all pre-cast. The gels come in either a 10-well or 15-well loading option. The gels come in a sealed plastic covering with a minimum amount of storage buffer so as to keep it moist during storage. Invitrogen generally sells these gels as a box of 10. However, trail samples or single gels are also available. The near neutral pH of 7.5 ensures stability of the proteins and the gel matrix during an electrophoretic run.

Upon assembly of these gels for an experiment, the important thing to remember is to remove the thin white sticker at the bottom in order to ensure current flow through the buffer system. The gel combs are also to be removed before loading the samples. It is always advisable to flush the wells with the running buffer before loading the samples in order to get rid of any residual acrylamide. This helps in uniformity of the running of the gel and sample resolution.

The gels are designed to perfectly fit in the Invitrogen’s XCell SureLock™ electrophoresis apparatus. The composition of these gels is based on a Bis-Tris system, which is probably superior in resolution to the traditional Tris-Tricine system. In the case of the Native PAGE gels, the samples are resolved based on the size/molecular masses and the migration of the samples is governed by Coomassie G-250 dye which binds to the proteins and confers a net negative charge without denaturing the proteins. The cathode buffer also contains G-250 which provides a continuous flow of G-250 into the gel; the gel in its original state doesn’t contain this dye. The addition of G-250 sample additive is a must when detergents have been employed in any step before the running of the gel. For non-detergent treated samples, the 4X sample loading buffer is sufficient enough, addition of G-250 is optional. All of the components: G-250 sample additive, 4X loading buffer, cathode buffer, anode buffer, etc. are available from Invitrogen directly. The whole system is also known as Blue-Native PAGE.

I have used the NativePAGE gels from Invitrogen extensively for my research. My protein complexes were big in size and thus, I mainly used the 3-12% gels. My molecular weight markers resolved sizes from 66 kDa to 1,236 kDa. My experience with this system has been extremely satisfactory and I have obtained very good resolution of the samples. After running the gel, I have used it in couple of different applications including Western blot, silver stain, drying and probing for autorad activity, and the system has performed will in all of these protocols. My standard running conditions have been at 105 V for 150 min. Typically, I have loaded 10 ug protein per lane and it has resolved very nicely. One thing which I have noticed is that loading too much protein is not well tolerated by the system. The other problem which I have faced while running these gels is the starting current. In my hands, the current supplied to the gels has ranged anywhere from 10 mAmps to 20 mAmps, and I have not been able to figure out what causes this variation. Also, the amount of current that flows through the electrophoretic system during the sample migration decreases as a factor of time as the gel runs.

The gel system works efficiently and I have not faced any major problems as such. The only downside is that all the reagents are recommended to be purchased by the supplier for best results, which sometimes might become a little budget issue.