Berglund:Western Blot: Difference between revisions
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label membranes so they don't get mixed up. Can be stored up to 1 yr.or longer!! <br> <br> | label membranes so they don't get mixed up. Can be stored up to 1 yr.or longer!! <br> <br> | ||
primary: 200µl 10% Tween 20<br> | [[primary: (2 gels)]] | ||
200µl 10% Tween 20<br> | |||
10µl MBNL Millipore<br> | |||
7µl GAPDH<br> | |||
10ml Odyssey blocking buffer<br><br> | |||
secondary 200µl 10% Tween 20<br> | [[secondary:(2 gels)]] | ||
200µl 10% Tween 20<br> | |||
0.7µl Mouse 800<br> | |||
0.7µl Rabbit 700<br> | |||
10ml Odyssey blocking buffer<br> |
Revision as of 10:26, 17 December 2013
MBNL Western
Cell Treatment
- Ripa Buffer (final concentrations)
- 25mM Tris HCl pH 7.6
- 150mM NaCl
- 1% NP-40
- 1% Na deoxycholate
- 0.1% SDS
- make in amber bottle, store at RT
- ****This is a 1.1x solution
- fc= 1x after addition of inhibitors
- 0.1M PMSF
- wear a mask when handling PMSF
- use 100% (200 proof) EtOH, make about 5ml of solution
- vortex to dissolve
- ****this is a 100x solution (sometimes viewed as 50x)
- store at -20
- stable for 1 month, may crash and have to be resuspended
- wear a mask when handling PMSF
- SigmaFast protease inhibitor pill
- Dissolve 1 pill into 10ml water
- store in fridge, it is never fully suspended, shake before using
- undissolved pills are good for 4 years
- dissolved pills are good for at least 2 weeks (good for 1 month)
- ***This is a 10x solution
- Dissolve 1 pill into 10ml water
- Check the plate for confluency
- take to lab, and prep the buffer
- 24 wells = 30 ml RIPA, 30µl of PMSF and 300µl of SigmaFast all on ice
- aspirate the media, do not wash cells, add 100µl Ripa complete to each well
- shake in cold for 10 minutes on speed 6
- place in eppendorfs and can be stored at -20°C over weekend
- or (and this has to be done before using to complete lysis) vortex in cold on speed 4 for 15 minutes
- spin at 12,000rpm for 15 minutes in cold
- transfer supernate to new tubes on ice and freeze at -20°C
Gel
- Pour 10% SDS protein gels (15 well combs)
- take 12µl of lysate and add 3µl of 5xSDS loading dye
- heat for 3 minutes
- spin down for .5 min
- load onto gels 12µl
- load 1µl of PAGE ruler prestained ladder
- run until dye is almost off
- rinse rig, open plates, cut off stacking gel
Western
- find the black and clear plate western sandwich cassette rig
- open like a clam and put into a dish of 10% methanol/1x transfer buffer
- place in order on the black side:
- sponge
- 2 pieces of whatman paper
- gel on of whatman
- (shiny side faces gel) nitrocellulose, 0.45µM (roll) no touch
- 2 pieces of whatman (roll)
- sponge
- clear side
- clamp it all down
- place in rig, black side to black side
- add ice pack
- fill with 10% methanol/1xtransfer buffer
- run in cold room for 1 hour at 500milliAmps
- dispose of buffer in container under sink
- pull rig apart
- rinse staining dishes with methanol, collect the flowthrough for proper disposal
- do the Ponceau stain
- replace stain into bottle and then take a picture of the gel
- put membrane in box shiny side up, destain in TBST (throw down drain)
- Block 1hr at RT with 5 ml or so of Odyssey blocking buffer you can reuse this for your 1°
Antibodies
primary are light stable
secondary are fluorescent and unstable, treat accordingly
Primary goes on for 1 hour to overnight
Secondary goes on for 1 hour
Wash post 1° and 2° with TBST: 1 min, 1min, 5 min, 10 min
always pour out of the same corner
handle only with special forcepts, never touch the membrane
label membranes so they don't get mixed up. Can be stored up to 1 yr.or longer!!
primary: (2 gels)
200µl 10% Tween 20
10µl MBNL Millipore
7µl GAPDH
10ml Odyssey blocking buffer
secondary:(2 gels)
200µl 10% Tween 20
0.7µl Mouse 800
0.7µl Rabbit 700
10ml Odyssey blocking buffer