esteemed researchers,
I am working on Mn2+ phos-tag gel for the first time as a young researcher, I have tried to optimise the gel several times to obtain clear unwavy bands and well separated bands. it has been a difficult task all together, to make it easier I will state the protocol I use here:
phos tag gel- 8%
separation gel:
water - 2.6ml, 1.5M TRIS ph8.8- 1.25ml, 10%sds- 50ul, 10mM MnCl2- 50ul, acrylamide- 1ml, 5mM phos tag(AAL-107)- 20ul, 10%APS- 25ul, Temed- 5ul
separation gel is top filled with saturated butanol-water, gel is washed with water after polymerisation.
Stacking gel:
water- 3.15ml, 0.5M tris ph6.8- 1.25ml, 10%sds- 50ul, acrylamide- 500ul, 10%Aps- 25ul, Temed- 5ul.
I work with TCA extracted proteins dissolved In Human urea(HU).
AFTER RUNNING GEL;
gel is soaked in 50ml sds running buffer containing EDTA (1:100 5mM EDTA)
gel washed in 50ml sds running buffer for 10 mins
transfer: pvdf briefly soaked in methanol.
results so far: multiple bands, wavy and not well separated.
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