I am doing 2D electrophoresis but not with IPG strip this time but simple PAGE run before 2D, strip is cut from PAGE gel and placed horizontally on second dimension gel.
Normally the articles that i read used smaller spacer width in the second dimension i.e first dimension run in 1mm spacer plate and second dimension run in 0.75mm spacer plate. But i do it oppositely i.e my first dimension gel is run in 0.75mm spacer plate and second dimension in 1mm spacer plate. I do it because my first dimension gel strip easily slides down the 1mm spacer plate in second dimension. I polymerize the second dimension gel first and then slide the first dimension strip onto it.
Now i have noticed oen thing that my two gels are not perfectly i contact with each other even though i overlay the gel and strip with stacking gel and then 0.5% agarose. and i think because of this i am getting a lot of horizontal streaking.
Can anyone please guide me regarding this. and if you have any video of such manual 2D methods please share it with me