3 replies to this topic

[kadamb]

I am running 2D gels and after doing silver staining i saw streaking like a column, starting from the top to the bottom, though there are many spots but i don't understand why streaking?, i am using DTT and IAA for equlibration buffer, does any of these can cause this or any idea what happens if equilibration time is prolonged. Or does excess amount of DTT or IAA  can cause these problems.
Thanks a lot

[Eric Jordens]

Dear kadamb,

I don't know if an excess amount of DTT or IAA might couse this streaking. I use the protocol form A. Görg and the only time I encounterd this streaking was when I forgot to add a 'low-current sample protein entry phase' in the 2nd demension part when using the flatbed system from APB. This is supposed to give the proteins time to enter the gel. I don't know if this applies for the system you are using.
This link to the trouble shooting section of A. Görg might help:
http://www.weihenstephan.de/blm....p13.htm

[kenny]

hope this not too late to reply your problem

you should check your working solution... make sure they are freshly prepared like equilibrium buffer with DTT and with IAA. make sure also you use deionized distilled water

[phdconsult]

salt - there is too much salt in your sample. Streaking is caused by abnormal electrolyte presence which messes up the electrophoresis field. Plan on loading sample dye by itself on both sides of the electrophoresis plate. This straightens out the front and improves run quality but of course the salt needs to be removed for getting good runs for the long pull.

GoodLuck

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