Protocol Online logo
Top : Forum Archives: : Immunology and Histology

Vertical bands - (Nov/24/2005 )

Hi,

Ive been running 8.5% tris-tricine gels recently and have found that surrounding my band of interest around the 50kDa molecular weight mark I get vertical bands/streaks inbetween each lane.
Anyone have any ideas?

Thanx

-pahl-

QUOTE (pahl @ Nov 24 2005, 08:59 AM)
Hi,

Ive been running 8.5% tris-tricine gels recently and have found that surrounding my band of interest around the 50kDa molecular weight mark I get vertical bands/streaks inbetween each lane.
Anyone have any ideas?

Thanx


You may be loading too much sample, try to reduce the amout of sample (5ug -40ug per lanes).
Or
Your samples contains too much salt, you may dialysis your samples to reduce the salt concentration.

I hope this may help.

-Minnie Mouse-

Thanx i'll give it a go!!

-pahl-

hi,

you might be running into one of several issues:

1.) your stacking gel may be shrinking when it polymerizes, causing the wells to become considerably more shallow than when you poured them. if you overload your sample, some of it may settle on the gel between each lane, which will then cause artifacts.

2.) keratin. this protein runs somewhere near that size range, so be sure to filter all of your solutions through 0.22┬Ám filter to remove dust and other particulate which has keratin (no, whatman paper doesn't count). also be sure to keep your work area very clean and use gloves when handling your samples.

3.) rinse the wells of your gel w/ a syringe first w/ water, then w/ your cathode buffer to remove any unpolymerized acrylamide. if present, this can bind to your proteins and cause slight mobility shifts.

i hope this helps.

QUOTE (pahl @ Nov 24 2005, 11:59 AM)
Hi,

Ive been running 8.5% tris-tricine gels recently and have found that surrounding my band of interest around the 50kDa molecular weight mark I get vertical bands/streaks inbetween each lane.
Anyone have any ideas?

Thanx

-johanski-