Western Blot



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 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

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