Eccles:Western Blotting

Western Blotting

SDS-PAGE

TG

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

TGS

100ml TG
5ml 10% SDS
895ml dH₂O

Resolving Gel

Reagents	8%	10%	12%
37.5:1 bis/acrylamide (30%)	2.67ml	3.33ml	4ml
1.5M Tris HCl pH 8.8	2.5ml	2.5ml	2.5ml
10% SDS	100uL	100uL	100uL
MQH2O	4.62mL	3.96mL	3.29mL
10% APS	100uL	100uL	100uL
TEMED	10uL	10uL	10uL

Stacking Gel

Reagents	5%
37.5:1 bis/acrylamide (30%)	1.67ml
0.5M Tris HCl pH 6.8	2.5mL
10% SDS	100uL
MQH2O	5.62mL
10% APS	100uL
TEMED	10uL

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

• Clean glass gel plates with 70% EtOH
• Make up resolving 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 MQH₂O to create straight edge
• Allow to set for 30mins
• Unclip gel from pouring stand and tip water 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 (if you leave stacking gel to set for longer the wells shrink)
• 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 15uL in 1x loading dye (3uL of 5x loading dye in 15uL) in PCR tubes
• Heat to 95degreesC for 5min
• Spin samples (13000rpm, 1min)
• Load immediately
• Load marker of choice following manufacturer's instructions
• Run gel at 120V for a suitable length of time to view your protein(s) of interest. 1.5h runs the shortest marker (10-20kD) to the end of a 10% gel

Western Transfer

1x Blot Buffer

100mL TG
100mL 100% methanol
2.5mL 10% SDS
797.5ML dH₂O

For semi-dry transfer unit 100mL of transfer buffer is plenty.

10mL TG
10mL 100% methanol
0.25mL 10% SDS
79.8mL dH₂O

• Cut a piece of PVDF membrane the same size as your gel for the transfer (5x8cm² usually suitable) - WEAR GLOVES when handling the membrane
• Wash membrane in 100% methanol, 5min
• Wash membrane in 1x Blot Buffer, 5min
• To prepare gel for transfer, switch powerpack 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
• On semi-dry transfer unit place the masking sheet (transfer hole should be slightly smaller than your gel size, say a mm smaller on each side)
• Take 3 pieces of 5x8cm² or suitably sized blot paper and dip into 1x Blot Buffer and place onto transfer hole of masking sheet (refer to semi-dry transfer unit diagram)
• Place PVDF membrane onto stack. Work quickly so membrane doesn't dry but simply pour a bit of Blot Buffer onto membrane if you need more time
• Place gel onto PVDF membrane
• Make sure there are no air bubbles between gel and membrane. Roll out if necessary
• Add 3 more sheets of Blot Buffer dipped blotting paper
• Tip a bit more 1x Blot Buffer over whole transfer stack just to moisten the transfer stack itself
• Place lid on semi-dry transfer unit evenly
• Plug connection in from lid to base
• Connect base to power pack
• Set power pack to LOWEST possible setting
• Switch on
• Desired current is 0.8mA per cm² so for a 5x8cm² gel you want 32mA. The current starts off higher than this but does gradually drift down to the desired current.
• After an hour the current will need turned up a notch to maintain 32mA
• Transfer proteins from gel to membrane for 2 hour especially if you are looking at proteins 80kD and larger to ensure complete transfer. Felix successfully detected pax2 after a 1 hour transfer using the semi-dry transfer unit. Grace has coomassied a gel following semi-dry transfer and found after 2 hours the largest proteins do not completely transfer (200kD). I have observed that all of the marker bands transfer across completely after 2 hours.
• Once your transfer is complete disassemble stack and rinse membrane in MQH₂O for 5mins.
• If you wish to test the quality of the transfer, stain your gel as desired at this point

Immunostaining

10x TBS

24.2g Tris
80.6g NaCl
900mL dH₂O
pH to 7.6 with HCl

TBST

100mL 10x TBS
1mL Tween 20
899mL dH₂O

5% LFM in TBST

2.5g low fat milk powder
Make up to 50mL with TBST

0.1% LFM in TBST

0.4mL 5% LFM in TBST
19.6mL TBST

• Block membrane in 5% LFM in TBST for a minimum of 1h (do whatever is convenient, eg: overnight). (I do this in a pipette tip box lid using the whole 50mL of 5% LFM in TBST on the shaker)
• Wash membrane briefly in TBST
• Incubate membrane with primary antibody at desired dilution and time in 0.1% LFM in TBST (I do this in a 50mL tube using 10mL of 0.1% LFM in TBST on the ROTO-TORQUE rotator)
• Wash membrane 3 x 5min in TBST (done in pipette tip box lid using 20mL of TBST for each wash on the shaker)
• Incubate membrane with secondary antibody at desired dilution and time in 0.1% LFM in TBST
• Turn developer on (needs 15min warm up time)
• Wash membrane 3 x 5min in TBST
• Place membrane protein side up on clean acetate sheet
• Pipette 2mL of a 1:1 mix of Pierce Supersignal West Pico enhancer and peroxide solutions onto membrane (make sure spread across whole membrane)
• Incubate for 5min
• Use blot 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...