Preparing an Agarose Gel

(1) 200 ml of 1X TAE into measuring bottle.

(2) add 3 g Agarose

(3) microwave until dissolved

(4) add 13 ul ethidium bromide

Preparing a 2% Agarose Gel

(1) 2g agarose in 100 ml of 1x TEA Buffer

(2) boil    (3) cool slightly    (4) 5 ul ethidium bromide     (5) pour gel

An SDS Protocol

(1) mix 4 ul unknown (8ug), 6 ul water and 10 ul NUPAGE sample buffer (2x) (2 ul mercaptoethanol/100 ul sample)

(2) boil for 5 minutes

(3) fill upper chamber with 200 ml 1x MES running buffer containing 500 ul antioxidant.

(4) fill lower buffer chamber with 1x MES running buffer

(5) load 10 ul SDS-PAGE standards (Novex Mark-12) into one well

(6) load 10 ul known into well.

(7) Run at 200 V for about 45 min-1 hour (untill dye front reaches the bottom)

(8) Remove gel from cassette and place in a container with 100 ml DI water

(9) microwave on high for 1 minute

(10) shake gently for 2 min using shaker

(11) Discard water and repeat 2 times (100 ml water/microwave/shake)

(12) discard water after final wash and add about 25ml Simply Blue Safestain

(13) microwave on high for 1 minute

(14) shake until bands become visible

(15) discard stain and destain by shaking in 100 ml DI water

(16) determine molecular weight of unknown

Isoelectric Focusing

(1) Mix 4 ul unkown (8 ug), 6 ul water, and 10 ul IEF sample buffer (2x)

(2) dilute IEF cathod buffer 1:10 before use and de-gas for 10 minutes under vacuum

(3) fill upper buffer chamber with cathode buffer

(4) dilute IEF anode buffer 1:50 before use and fill lower buffer chamber

(5) load 10 ul IEF markers into one well

(6) load 10 ul known into well

(7) rune the gel 100 V for 1 hour, then 200 V for 1 hour then 500 V for 30 min

(8) remove gel from cassette and fix for 15 min with IEF gell fixative (12% TCA w/ 3.5% sulfosalicylic acid)

(9) rinse gel with 100 ml DI water 3 times for 5 minutes. discard last rinse

(10) stain gel with simply blue safestain for 1 hour at room temp. discard stain

(11) wash gel with 100 ml DI water for a minimum of 1 hour

(12) determine isoelectric point of unkown