5 replies to this topic

[shilpeejnc]

my purified protein is at ph 6.8 and another at 8.when I rin it on the gel they diffuse a lot.the band is wider.but o the same gel with same size wells,BSA loaded gives fine bands with separate wells.what could be the reason for it.

[rkay447]

Are you running the same amount of protein in each well?  When I run a sample with lots of protein, it tends to spread out and squeeze the lanes around it.  Also, what size is your purified protein?  My bands tend to get broader in lower percentage or at the bottom of a gradient gel.  If you are running a lot of protein, it may help to load a lot of sample buffer in the surrounding lanes.

[protolder]

Hola, It could be due at high salt concentration. For instance, when you run the elution fractions of an ion exchanger, as much higher salt concentration much wide bands . Good luck

[shilpeejnc]

rkay447, on 13 September 2010 - 11:27 AM, said:

Are you running the same amount of protein in each well?  When I run a sample with lots of protein, it tends to spread out and squeeze the lanes around it.  Also, what size is your purified protein?  My bands tend to get broader in lower percentage or at the bottom of a gradient gel.  If you are running a lot of protein, it may help to load a lot of sample buffer in the surrounding lanes.

size of my protein is 55 kda, 12% gel.

[shilpeejnc]

protolder, on 14 September 2010 - 09:27 PM, said:

Hola, It could be due at high salt concentration. For instance, when you run the elution fractions of an ion exchanger, as much higher salt concentration much wide bands . Good luck

how does salt affect the migration.MY ion exchange has .5M nacl and NINTA elute ha 300 mM imidazole. When they move on gel they make a curve.

[protolder]

Hola,  Probably 0.5 M NaCl makes diffuse the band but you have to see it aplying in the gel the sample without salt before ion exchanger.Imidazole at these concentration doesn´t affect to the diffusion. Have a nice day