SDS tracking dye running stragely - (Nov/13/2005 )

hey guys!

i'm having trouble with the proteins in my gel. i'm always having vertical streaks (from the side of the wells) while running SDS-PAGE. i also noticed that some of my tracking dye is being left from the top forming an inverted tooth with two roots at the top. in this case, i end up having side streaks along the side of the wells after i stain the gel with coomassie blue. someone told me that my sample might be precipitating while running or just when i loaded my samples. but i don't know how to troubleshoot it. i am warming up the running buffer to 37C but still getting the same vertical streaks. the sample should run along a horizontal line...not leaving any of the dye/sample right??

could anyone help me about this??

ann

---

I would say that it's due to your stacking gel, they could be also a role of the DNA if you load too much proteins extract and if it's very viscous.

Anyway that's how I proceed
Poor the running gel 10 to 15 %Acryl/Bis
cover the interface with isopropanol 50% in water let polymerise at least 30mn
dump out the isopro mix wash with distilled water and dry well the interface (with air stream or whatman paper slices) before to pour down the stacking gel 5%Acry/Bis

Wash well your comb to remove any traces of polymerized acrlyamide

let polymerise at least 30mn (always good to keep a bit of the solution in a tube as a contrôl)

Remove the comb while washing with disstilled water it facilitates the removal of the comb

Flush out well your wells with first distilled water and then with running buffer (best done with a syringe and a neddle flushing one by one the wells)

Hope it helps !

pesji

---

Could I also add, too much glycerol in the sample makes a pattern like that as well?

have you tried some freshly made sample buffer? perhaps the glycerol was too high in the last batch or something like that.

---