Cell Transformation Group:Protocols/Protein

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Harvesting Cell Lysates for Western Blotting (NEW METHOD)

(Takes about 1-1.5 hours)

1. Harvest cells and supernatant into a Falcon tube by trypsinisation
2. Use aliquot of supernatant to collect remaining cells in flask or 6-well plate. Transfer to appropriate tube
3. Spin for 5-10mins @ 250g (turn on 4°C centrifuge)
4. Discard supernatant into Virkon
5. Resuspend cells in 1ml of PBS and transfer to a tabbed, eppendorf tube
6. Remove a 20µl aliquot for counting
7. Spin for 10 mins @ 3000rpm/4°C (get ice)
8. Discard supernatant and resuspend in appropriate volume lysis buffer (+1xComplete) to resuspend at 2 x 104 live cells/µl or other useful concentration depending on expt
9. (Can do this now or later) Lyse cells for 30mins on ice, mix, spin cell debris down, transfer supernatant to labelled eppendorf
10. Store at –20°C

Cell Lysis Buffer
Concentration Reagent
10mM Tris HCl pH 8.0
150mM NaCl
1% NP40
0.1% SDS
1x Complete protease inhibitor

Add Complete just prior to use
Nb – Complete is stored at 25x concentration

40μl 25x to 1ml volume
2μl 25x to 50μl volume

Western Blot

Dilute samples Spin samples to pellet DNA. Dilute samples in appropriate amounts of DSL. Boil for 5mins, spin (fast) and then load. If using Biotinylated marker, aliquot out into new eppendorf, dilute in running buffer (1:50), then boil.

Run 10% mini gel for blotting 1. Clean gel plates and spacers with H2O and 70% EtOH. Assemble plates in gel former. 2. Pour 10% separating page (3.3ml/gel BIO RAD; 6ml/gel HOEFFER). 3. Layer with saturated butanol (or H2O) to removes bubbles. Leave to set 20-60mins. 4. Remove butanol/H2O by rinsing well with dH2O from water bottle. Use small pieces of filter paper to remove excess H2O taking care not to touch gel. 5. Pour 5% Stacking Gel. Insert comb and leave to set about 10-30mins. Rinse wells thoroughly with H2O from water bottle (gently). 6. Place gels in tank with wells facing in. (place plastic block with small glass plate attached on opposite side if only 1 gel is being run – BIO RAD) 7. Spin samples and load 15μl per well for 10 well comb (10μl/well for a 15 well comb). Load 10μl of appropriate marker (refer notes if using chemiluminescent detection) 8. Top tank up with 1 x SDS Running Buffer. Run at 120 volts for 1 – 1.5hrs (until dye front is in line with bottom gasket). For the Hoeffer system this may take 3hrs – be warned.

Blot (use transfer cell unit and cooling coil – BIO RAD system) 1. Set up transfer unit with cooling coil and add most of Blotting/Transfer Buffer. 2. Pre soak gels in Transfer Buffer. Soak white transfer pads and 4 sheets of filter paper in buffer in transfer unit. Pre-soak membrane filters in Analar Methanol. 3. Assemble blot on BLACK SIDE of transfer tray.

    layer :-  	1/ white transfer pad
                	2/ 2 sheets of pre-soaked filter paper
                 	3/ gel(s)
                   	4/ membrane(s)
                   	5/ 2 sheets of filter paper (smooth out bubbles with 10ml pipette)
                   	6/ white transfer pad

4. Close assembly tray. Slide handle across. Line up spacers. 5. Put into transfer tank with black side to black back. 6. Run overnight at 15 volts/100 milliamps 7. Check H2O pressure through coil as pressure sometimes drops off. 8. Take 1° and 2° antibodies from freezer to fridge for following day.

Blot (using HOEFFER system) 1. Open up transfer unit and assemble blot and gel sandwich(es) on the black side. 2. Pre soak gels in Transfer Buffer. Soak white transfer pads and 4 sheets of filter paper in buffer in transfer unit. Pre-soak membrane filters in Analar Methanol. 3. On BLACK SIDE of transfer unit:

    layer :-  	1/ coloured transfer pads (2 or 3)
                	2/ 2 sheets of pre-soaked filter paper
                 	3/ gel
                   	4/ membrane
                   	5/ 2 sheets of filter paper (smooth out bubbles with 10ml pipette)
                   	6/ coloured transfer pads (2 or 3)
                  	7/ repeat steps 2-6 for another gel

4. Click unit closed. Fill with transfer buffer. Tap sides to remove extra bubbles. 5. Assemble unit. Add cold H2O to tank (up to max level). Top up transfer buffer in unit. Transfer overnight at 15V (or for 2h at 25V if wanting to block overnight).