Hello.
I will conjugate 2 proteins by chemical crosslinking.
Then, I want to check whether protein are conjugated well or not, via NATIVE-PAGE (Conjugated proteins will be 200kDa~ 1000kDa in my assumption).
But, I don't have any experiences on NATIVE-PAGE or even SDS-PAGE.
Here, I have questions.
(1) Conjugated proteins will be 200kDa~ 1000kDa in my assumption. I don't know the gel composition. Could you let me know the gel composition? Papers or website that I can find information are welcomed..
(2) In my lab, there are equipment for PAGE. Gel size must be 8cm x 11cm for this equipment.
If I use 8cm x 8cm gel (commercially available), will it be a problem?
(3) I have searched gel composition of NATIVE-PAGE gel.There are lots of information about proper gel composition depending on protein size in case of SDS-PAG. But I couldn't find information about proper gel composition depending protein size for NATIVE-PAGE. Please let me know gel composition depending on various protein size for NATIVE-PAGE. Any information will be helpful to me.
Thank you.
Dear Sung-gil Hong,
if you cross-link your sample it should be stable enough to survive SDS PAGE. If you cross-link with sulfo-reactive cross linkers do not forget to omit reducing agents from the loading dye.
If, for any other reason, you still prefer Native PAGE you can read through the paper attached.
For your estimated size I would recommend a gradient gel that resolves several complexes of different sizes. Once you know size of your cross-link you could switch to regular gels. For 200-1000 kDa we normally use 4-13% PAGE. You can also pour gradient SDS PAGE.
If there is any trouble to prepare a Native PAGE in your lab you could buy one. Commercially available ones are compatible with regular power supplies etc.
Article Native electrophoretic techniques to identify protein-protei...
If you are buying prepared gels, they should be the same size as the apparatus, and they should be made for use with the apparatus. For such large molecules, a very low percentage gel is needed.
here your chemical cross linker must be stable to pass through the pore. Obviously for 200-1000kDa, 4-10% page-gel is preferred to have large porosity and less run time to save the protein sample from diffusion from running track..
If native-page does not work, why you not try to separate both the protein separately?
- Badges
- Science method