Eccles:LS MITF westerns

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MITF western blot protocol (Zymed C5+D5 antibody)

• 1Total cell Lysate

Method KEEP ALL REAGENTS ON ICE TO USE • Wash cells with ice cold PBS • Harvest cells into 15mL tube by typsinisation as usual • Spin at 250g for 5min • Aspirate off supernatant • Wash with PBS, spin. Prepare RIPA complete buffer place on ice with tubes • Resuspend cells at 10,000cells/ul in ice cold complete lysis buffer • Leave on ice for 30 minutes or store at -20C. Cool centrifuge • (On thawing) spin cells at 13.2 Krpm at 4C for 20mins • Aliquot supernatant into ice cold eppendorfs and store at -80 degrees C (20-40 ul aliquots usually helpful). NB transfer 4 ul into tube for BCA assay.

RIPA buffer (without protease inhibitors), store at 4°C

1 M Tris.Cl pH8.0 0.5ml 0.05M 1 M NaCl 1.5ml 0.15M 10% NP-40 1ml 1% Sodium deoxycholate 50mg 0.5% 10% SDS 100ul 0.1% MQ H2O 6.9ml Total 10ml

Cell lysis buffer (only prepare what you need)

20X Complete protease inhibitor 50ul 0.1 M PMSF (available in freezer) 10ul 100 uM NaOrthovanodate (available in freezer) 10ul RIPA buffer 930ul Total 1000ul

• 2 PAGE Gels

Buffers: TG X10 15.14g Tris (0.25M) 72g Glycine (1.9M) Make up to 0.5L with dH2O

TGS 100ml TG 5ml 10% SDS 895ml dH2O

5x Loading Dye 4mL 1M Tris pH 6.8 (0.4M) 5mL Glycerol, 100% (50%) 1g SDS (10%) 0.025g Bromophenol blue (0.25% w/v) 0.30g DTT (3% w/v) 1mL dH2O

Method • Clean glass gel plates with 70% EtOH • Make up resolving gel 10% and stacking gels minus APS and TEMED • Assemble gel plates onto stand • Add APS and TEMED to resolving gel mix • Pour resolving gel • Layer resolving gel with 1% SDS to create straight edge • Allow to set for 30-60mins • Unclip gel from pouring stand and tip SDS off resolving gel • Blot away excess moisture with blotting paper (don't contact gel) • Add APS and TEMED to stacking gel mix and pour stacking gel • Insert comb and top stacking gel up if necessary to ensure wells will be full depth • As soon as stacking gel is set (10-15min) remove gel from pouring assembly and rinse glass plates so there are no bits of gel stuck to the glass plates • Remove comb and rinse wells with dH2O • Flick water off taking care not to squeeze the gel • Rinse wells another two times • Assemble gel in running tank • Pour TGS running buffer into centre of assembly and check for leaks • If assembly is not leaking buffer add enough TGS to submerge bottom of gel by 1-2cm • If assembly is leaking buffer fill tank with TGS running buffer • Prepare protein lysate in a final volume of 20uL in 1x loading dye (4uL of 5x loading dye in 20uL) • Heat to 95degreesC for 5min (heating block) • Spin samples (13000rpm, 1min) place on ice 1 minute then • Load immediately • Load marker of choice following manufacturer's instructions (use 2ul Magic marker, 5ul Rainbow marker) • Run gel at 100V for about 1.5hr, dye front should be at the bottom of the gel

• 3 Western Transfer

1x Blot Buffer 100ml TGx10 200ml 100% methanol 10ml 10% SDS 690ml dH2O • Use nitrocelluose membrane - WEAR GLOVES when handling the membrane • Wet membrane & filter paper in 1x Blot Buffer, 5min • To prepare gel for transfer, switch power pack off • Tip off buffer and remove gel from running assembly • Prise open glass sandwich with provided wedge • Use narrow tip of wedge to slice stacking gel off and remove. • Put resolving gel into 1x Blot Buffer while prepare for transfer • Assemble blot on BLACK side of transfer tray o Layer 1. 1 White transfer pad o 2. 2 sheets filter paper o 3. Gel o 4. Nitrocellulose o 5. 2 sheets filter paper o 6. Make sure there are no air bubbles between gel and membrane. Roll out if necessary o 7. 1 White transfer pad • Close assembly tray, slide handle across & line up spacers • Put into transfer tank with BLACK SIDE TO BLACK BACK • Place transfer buffer in the unit • Put assembly into unit & run at constant amps 225mA for 1 ½-3 hours (NB if you run 2 gels change the gels around half way through & add fresh buffer) • When finished dissassemble • To visualize tranfer place membranes in 0.5% ponceau red/3-5% acetic acid & if the transfer is OK proceed to immuno.

1. 4 Immuno

Use Western Breeze kit & follow their instructions.

• Block membrane for 30 minutes at RT • Wash membrane 2 x 5min in water • Incubate membrane with primary antibody MITF (Zymed) diluted 1/1000 over night at 4C. • Wash membrane 4 x 5min in wash solution • Incubate membrane with secondary antibody 30 minutes at RT • Turn developer on (needs 15min warm up time) • Wash membrane 4 x 5min in wash solution • Rinse membrane 2 x 2min in water • Place chemiluminescent substrate on clean acetate sheet • Place the membrane on, protein side down, & make sure membrane is covered with the solution by pulling the membrane up & down • Use blotting paper to blot off excess solution • Place acetate sheet over membrane and smooth out air bubbles • Place membrane sandwiched between acetate sheets into developing cassette and head to developing room

Place film against membrane and develop...