Eccles:LS MITF westerns

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MITF western blot protocol (Zymed C5+D5 antibody)

Total 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 H₂0 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

PAGE Gels

TG X10

15.14g Tris (0.25M)
72g Glycine (1.9M)
Make up to 0.5L with dH₂0

TGS

100ml TGx10
5ml 10% SDS
895ml dH₂0

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 dH₂0

• 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

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
• Layer 1. 1 White transfer pad
• 2. 2 sheets filter paper
• 3. Gel
• 4. Nitrocellulose
• 5. 2 sheets filter paper
• 6. Make sure there are no air bubbles between gel and membrane. Roll out if necessary
• 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.

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...