Western Blot
Protocol:
I. Preparing Samples
· Label tubes for samples, get bucket of ice
· Aspirate off old media
· Add 3mL 1X PBS and aspirate
· Add 500uL MAPk Buffer and scrape (this amount can be adjusted depending on confluency of your plate)
· Transfer to 1.5mL tube
· Spin 12,000rpm for 10min.
· Transfer supernatant into 1.5ml tube
· Freeze @ -20oC if not running gel right away
II. Invitrogen midi pre-made gels.
III. Transfer on Invitrogen iBlot apparatus.
Protein of interest <30kDa, use program P3 for 6 minutes
Protein of interest >30kDA, use program P0 for 7 minutes
IV. Blocking
· Casein solution; already made (stored @ 4oC)
· Put membrane in plastic box
· Cover membrane with solution
· Rock @ 4oC for 1 hour (or longer)
V. Primary Antibody
· Dilute accordingly in 5-15mL of Casein+Tween ( i.e. 1:1000 of B-actin in 10mL = 10uL of B-actin)
· Add 1o Ab directly to blocking solution
· Rock @ 4oC O/N (or 1-2 hour minimum)
o Can store primary antibodies @ 4oC and re-use
* you can look in the Antibody notebook for more info.
VI. Secondary Antibody
· Save the primary antibody and label tube with antibody, secondary, any phosphorylated residues, date, and numbers of use.
· Wash membrane 4 x 5 min. in TTBS in carboy @ RT, rocking
· Dilute 2o Ab in casein + tween
· 2˚Ab dilution 1:15,000 = 15ml casein + tween and 1ul secondary antibody
· Rock at room temperature for 1 hour and prepare from light.
XIII. Wash
· Wash membrane w/ TTBS 4 x 5 min.; can be left in the last wash for extended period of time and protect from light.
XIV. Develop Using Odyssey
· Save blot if stripping and re-probing, otherwise discard
o Label your film with antibodies used, concentrations, date, ladder, samples, etc.
If stripping blot the following day:
Put in TTBS @ 4oC O/N (no rocking).
Stripping a Blot:
Place blot in odyssey stripping solution and shake for 20min at 37C.
Wash 4 x 5min with TTBS, block with casein for 1 hour
Add 1o AB and rock @ 4oC O/N … proceed from Step VI
Solutions
MAPKinase Lysis Buffer (250mL)
12.5mL 10% Triton-X-100
2.5mL 0.5M B-GP
500uL 1M MgCl
1.25mL 20mM Na3VO4
1mL 250mM EGTA
250uL 1M DTT
500uL 500X Leupeptin
500uL 500X Aprotinin
208.5uL H20
250uL 100X PMSF
10X PBS (1L):
80g NaCl
2g Kcl
14.4g Na2HPO4
2.4g KH2PO4
pH buffer to 7.4
1X PBS (1L):
100mL 10X PBS
900mL dI H20
5X Loading Dye (100mL):
3.78g Tris
25mL 25% glycerol
pH to 6.8 (always before adding SDS)
10g SDS
5mg Bromophenol blue
Aliquot into 7.5mL and store @ -20oC. Add 2.5mL B-Me when ready to use.
Johnson’s 10X Running Buffer (4L):
121.1g Tris base
576.7g Glycine
pH to 8.63
40g SDS (never use pH meter with SDS!)
bring up to 4L
Johnson’s 1X Running Buffer (4L):
400mL Johnson’s 10X Running Buffer
3600mL dI H20
TTBS:
3800mL H20
200mL 20X TBS
5mL Tween
1
LAW
June 13, 2012