Berglund:Western Blot: Difference between revisions
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'''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 | |||
[[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''' | |||
1 Check the plate for confluency | |||
2 take to lab, and prep the buffer | |||
3 24 wells = 30 ml RIPA, 30µl of PMSF and 300µl of SigmaFast all on ice | |||
4 aspirate the media, do not wash cells, add 100µl Ripa complete to each well | |||
5 shake in cold for 10 minutes on speed 6 | |||
6 place in eppendorfs and can be stored at -20°C over weekend | |||
7 or (and this has to be done before using to complete lysis) vortex in cold on | |||
speed 4 for 15 minutes | |||
8 spin at 12,000rpm for 15 minutes in cold | |||
9 transfer supernate to new tubes on ice and freeze at -20°C | |||
'''Gel''' | |||
1 Pour 10% SDS protein gels (15 well combs) | |||
2 take 12µl of lysate and add 3µl of 5xSDS loading dye | |||
3 heat for 3 minutes | |||
4 spin down for .5 min | |||
5 load onto gels 12µl | |||
6 load 1µl of PAGE ruler prestained ladder | |||
7 run until dye is almost off | |||
8 rinse rig, open plates, cut off stacking gel | |||
'''Western ''' | |||
1 find the black and clear plate western sandwich cassette rig | |||
2 open like a clam and put into a dish of 10% methanol/1x transfer buffer | |||
3 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 | |||
4 place in rig, black side to black side | |||
5 add ice pack | |||
6 fill with 10% methanol/1xtransfer buffer | |||
7 run in cold room for 1 hour at 500milliAmps | |||
8 dispose of buffer in container under sink | |||
9 pull rig apart | |||
10 rinse staining dishes with methanol, collect the flowthrough for proper disposal | |||
11 do the Ponceau stain | |||
12 replace stain into bottle and then take a picture of the gel | |||
13 put membrane in box shiny side up, destain in TBST (throw down drain) | |||
14 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 | |||
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: 200µl 10% Tween 20 | |||
(2 gels) 10µl MBNL Millipore | |||
7µl GAPDH | |||
10ml Odyssey blocking buffer | |||
secondary 200µl 10% Tween 20 | |||
(2 gels) 0.7µl Mouse 800 | |||
0.7µl Rabbit 700 | |||
10ml Odyssey blocking buffer |
Revision as of 10:03, 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
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
1 Check the plate for confluency 2 take to lab, and prep the buffer 3 24 wells = 30 ml RIPA, 30µl of PMSF and 300µl of SigmaFast all on ice 4 aspirate the media, do not wash cells, add 100µl Ripa complete to each well 5 shake in cold for 10 minutes on speed 6 6 place in eppendorfs and can be stored at -20°C over weekend 7 or (and this has to be done before using to complete lysis) vortex in cold on speed 4 for 15 minutes 8 spin at 12,000rpm for 15 minutes in cold 9 transfer supernate to new tubes on ice and freeze at -20°C
Gel 1 Pour 10% SDS protein gels (15 well combs) 2 take 12µl of lysate and add 3µl of 5xSDS loading dye 3 heat for 3 minutes 4 spin down for .5 min 5 load onto gels 12µl 6 load 1µl of PAGE ruler prestained ladder 7 run until dye is almost off 8 rinse rig, open plates, cut off stacking gel
Western 1 find the black and clear plate western sandwich cassette rig 2 open like a clam and put into a dish of 10% methanol/1x transfer buffer 3 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 4 place in rig, black side to black side 5 add ice pack 6 fill with 10% methanol/1xtransfer buffer 7 run in cold room for 1 hour at 500milliAmps 8 dispose of buffer in container under sink 9 pull rig apart 10 rinse staining dishes with methanol, collect the flowthrough for proper disposal 11 do the Ponceau stain 12 replace stain into bottle and then take a picture of the gel 13 put membrane in box shiny side up, destain in TBST (throw down drain) 14 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 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: 200µl 10% Tween 20 (2 gels) 10µl MBNL Millipore 7µl GAPDH 10ml Odyssey blocking buffer
secondary 200µl 10% Tween 20 (2 gels) 0.7µl Mouse 800 0.7µl Rabbit 700 10ml Odyssey blocking buffer