Hi all, I used pngase f to release glycan from transferrin. here is my protocol.
1. I prepared storage buffer for the pngase f without glycerol and aliquot them into different vial
2. I took 10 microgram of protein and denatured @ 100°C for 10 min
3. added Np40
4. then glycobuffer
5. pngase f added in the conc of 5mU, 50mU, 0.5U, 5U, 25U, 50U and incubated for 16 hrs.(1:10 v/v) total reaction vol 50 microlitre
6. I took 20 microlitre from that and prep for loading into sds page.
Here is my result. is my pngase f showing activity? or it looses activity because of no glycerol used in the buffer?
Lane 1 - standard
Lane 2 - without enzyme
Lane 3 - precipated after incubation
Lane 4 - 50U
Lane 5 - 25U
Lane 6 - 5U
Lane 7 - 0.5U
Lane 8 - 50mU
Lane 9 - 5U
You should use a lower percentage acrylamide gel in order to improve the ability to distinguish between different molecular weights around that of your protein. If the protein is glycosylated with a large amount of carbohydrate, you should be able to see the band shift to a lower position on the gel after PNGase treatment.
I ran at 12.5%. I will reduce my %. And Yes, we are expecting mobility shift. After this confirmation only, we are planning to do mass spec based glycan analysis. Thanks for your suggestion
Adam B Shapiro, I ran in 10% gel and got the clear separation of band. Now my another question is, everytime I run the gel, Dye front is moving in slanting fashion but at the end, I'm getting proper brand. All the buffers and chemical prepared for the gel preparation are common use in our lab. This case is happening in my setup only. What could be the reason? Why my gel is running slanty.
It could be a problem with the gel box, such as a leak, so that the electric field is not uniform. Or, it could be a big difference in the ionic strengths of the samples from well to well.
Adam B Shapiro , could you explain how ionic strength in the each well will differ? I found out that I was making mistake in loading my sample volume. I was concern about my concentration not the volume. Later that, I got some clear straight line. And I want to know how ionic strength affect the dyefront movement. could you please explain?
I don't know the reason for it, but I have observed that large differences between adjacent samples in the ionic composition can cause artifacts in SDS-PAGE. In particular, a high-salt sample (for example, a resuspended ammonium sulfate precipitate) will tend to spread horizontally into adjacent lanes.
- Badges
- Science topic