Protein Immunoblotting- An Introduction to Western Blotting

1) Eelectrophoretic separation of protein or of nucleic

acid fragments in the sample

2) Transfer to and immobilization on a matrix.

3) The probe is added to the matrix to bind to the target

molecules.

4) Any unbound probes are then removed.

5) Visualization of bound probe

Western blot Protein Detection

• Detects proteins and estimates their molecular

weight.

• Detects changes in phosphorylation and lipid

modifications.

• Used to detect changes in protein expression.

Sample Preparation

SDS-PAGE (Protein Electrophoresis)

Electro-transferring

Immunoblotting

• Major components of the sample loading “buffer”

– SDS

– DTT

– Tracking dye

– Glycerol

– Protease inhibitor

Western Blot Visual Protocol: Phase 1: Sample Preparation

• Sodium dodecyl sulfate (SDS)

• Tris buffer (either glycine or tricine)

• Acrylamide and NN-bis-acrylamide– Forms gel matrix

• TEMED– Catalyst for polymerization (produces free radials from APS)

• Ammonium persulfate (APS)– Source of free radials for polymerization

Could purchase pre-cast gels if you have the money.

Ingredients in Gel

Stacking (concentrating) gel4% acrylamide

0.5M Tris-H+Cl-, pH 6.8, 0.1%

SDS

Resolving (separating) gel10% acrylamide (36.5:1,

acryl/bis)

1.5 M Tris-H+Cl-, pH 8.8, 0.1%

SDS

Running buffer0.25 M Tris base; 1.92 M

glycine, pH 8.3; 1% SDS

Resolving gel

Stacking gel

Discontinuous system

How to Make an SDS-PAGE gel

Western Blot Visual Protocol: Phase 2: Protein Electrophoresis

• uses an electric current to pull proteins from the gel

into the PVDF or nitrocellulose membrane

• Transfer buffer contains: Tris, Glycine, and methanol

but no ions.

• Nitrocellulose membranes are cheaper than PVDF,

but are far more fragile and do not stand up well to

repeated probings

• Western blot transfer can be done in wet or semi-

dry conditions

• Equilibrate gel in transfer buffer

in separate tray.

• Equilibrate filters and sponges

in transfer buffer.

• PVDF membranes must be

soaked in methanol, before

equilibration in transfer buffer.

Nitrocellulose membranes are

soaked directly in transfer

buffer

Mount transfer sandwich in

blotting chamber which already

contains transfer buffer

Western Blot Visual Protocol: Phase 3: Membrane Transfer

• Washing (TBS-T)

• Blocking

• Incubation with Primary antibody

• Washing (TBS-T)

• Incubation with secondary antibody

• Blocking reduces

nonspesific binding of

antibody (primary or

secondary) to protein or

membrane

• Too little => high

background

• Too much reduces the

signal

• Incubation time:

– 1-2 hrs at RT with shaking

• Blocking agents

– Fat free dry milk

– Bovin serum albumin

– Casein

– Gelatin

– Hemoglobin

– Ovalbumin

• Buffer:– PBS, phosphate buffered saline, pH

7.5-8.0

– TBS, TRIS-buffered saline, pH 7.5

• After blocking membrane, add antibodies in to blocking solution (ie 5% milk).

• Incubate overnight at 4oC or 2 hours at room temperature.

• Buffer: PBS w/Tween 20 or TBS w/Tween 20

– TW20 concentration must be determined for each antibody and

antigen

– Usually 0.01-0.2%

• Time: Number of washes and duration of each wash

must be determined in each case

– Usually 3X5 min

– Use large buffer volume: 50-100 ml for 8X10 cm membrane

– Incubation with vigorous shaking

• Buffer: same as for primary antibody

• Dilution must be determined in each case

– Usually 1:1,000 - 1:100,000

• Incubation time must be determined in each case

– Varies from 5 min to 2 hrs

enzym

• Colorimetric detection

• Chemiluminescent detection

• Radioactive detection

• Fluorescent detection

Western Blot Visual Protocol: Phase 4: Immunoblotting