Protein Detection Methods & Western

Blots

1. Sodium Dodecyl Slufate (SDS)-Polyacrylamide Gel

Electrophoresis (SDS-PAGE)

• Visualize many proteins at once

2. SDS-PAGE combined with immunoblotting

(Western)

• Visualize one protein out of complex mixture

What is SDS-PAGE?A procedure to separate proteins and determine their

Molecular Weights.

Theory Behind Electrophoresis

• Charged molecules in an electric field behave in a

predictable manner.

• Positively charged molecules will move towards the

negative pole while negatively charged molecules

move towards the positive pole.

• Movement of any charged species through an electric

field is determined by it’s net charge, molecular radius

and the magnitude of the applied field, but not size.

How do we get proteins to separate by

size?

•Need to disrupt tertiary structure

•Make charge uniform

The importance of SDS

SDS is a negatively charged (anionic) detergent.

Coats proteins and disrupts secondary and tertiary protein structures by breaking hydrogen bonds and unfolding or denaturing protein.

‘Masks’ charge on protein by imparting a large negative charge so that all proteins act the same in regards to charge.

Prevents protein aggregation.

Prevents protein shape from influencing gel run. Size of protein determines migration in gel.

SDS linearizes proteins

•All proteins have the same mass to charge ratio

•Have the same molecular radius

•Thus, migration through a matrix is determined

by length which is proportional to molecular

weight

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SDS-PAGE: The Matrix

Free radicals

Transfer of electrons to acrylamide and

bisacrylamide; causing them to react

Amount of crosslinking, thus pore size and

consequent separation properties of the gel

can be controlled by varying the ratio of

acrylamide to bis-acrylamide

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SDS-PAGE: The buffer system

Stacking gel: 4%

Discontinuous Laemmli buffer system - buffer

in the gel and the tank are different

Resolving gel: range 5-20%

Gly

proteinsCl-

Proteins are concentrated into the narrow

zone between the Cl- and glycine fronts.

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Polyacrylamide concentration alters

resolution of protein migration

Uses of SDS-PAGE

• Determine protein size

• Identify protein

• Determine sample purity

• Identify existence of disulfide bonds

• Quantify amounts of protein

Detection of protein after SDS-PAGE

STAINING

Technique - The goal of staining is to bind a chromaphore to a

polypeptide

Not Quantitative - All stains are very qualitative, individual polypeptides

differ greatly in their ability to bind a stain

Low Reproducibility - Different staining techniques will not stain a

polypeptide consistently

Range - There is a small concentration range over which a particular

stains is useful

Non-Specific – Binds to majority of proteins; unable to determine if your

protein of interest is in the protein extract run on the gel

Protein dyes used for stainingStain Advantages Disadvantages

CoomassieBlue

Simple methodologyMS compatibleEasy to image

Very insensitive

Bio-SafeColloidalCoomassieStain

Simple methodologyMS compatibleMore sensitiveEasy to image

Less sensitive thansilver

Silver stain Very sensitiveEasy to image

Complex procedureIncompatible with MSNot quantitative

Sliver stainPlus

Very sensitiveEasy to imageMore MS compatible

than standard silverstaining

Complex procedureNot very MS

compatibleNot quantitative

Ruby stain Very sensitiveMS compatibleQuantitative over 3

orders of magnitudeSimple methodology

Difficult to image

Other Methods of Protein Detection

1. Labeling proteins with radioisotopes during

expression (e.g. 35S-methionine)

1. Western blotting (e.g. antibody against protein

of interest)

Comparative Proteomics Kit II:

Western Blot Module

Bio-Rad’s take on Western blotting!

Why Teach

Western Blotting?

• Powerful teaching tool

• Real-world connections (?)

• Laboratory extensions (?)

• Tangible results

• Link to careers and industry (?)

• Standards-based (?)

According to Bio-Rad

Why use Western blotting?

•Can specifically determine if your protein of

interest is present in a crude protein preparation

Transfer

Proteins from

the gel to the

nitrocellulose

membrane

60 minutes

200 mA

Blotting buffer

1x Tris glycine

with 20%

methanol,

0.1% SDS

Electric Current

Protein Transfer

Blocking

Buffer

Remove membrane from the blotting sandwich

and immerse in blocking solution

5% non-fat milk or 1% gelatin: Prevents the

primary antibody from binding randomly to the

membrane

Tris or Phosphate buffered saline (TBS or PBS):

Provides the correct environment (pH, Salt) to

maintain protein shape

0.025% Tween 20: non-ionic detergent that

prevents non-specific binding of antibodies to the

membrane; TBS-T or PBS-T when added to buffer

Blocking membrane before addition of antibody

Antibodies used for Western

• Molecule of interest is injected into primary animal model

• Animal makes antibodies against the molecule

• Antibodies are purified (primary antibody)

Addition of the 1o antibody•Discard blocking solution

•Pour primary antibody (in TBST) onto the membrane

and gently rock for 30 minutes

•Primary antibody will bind to the histidine tag

•Add 50ml of wash (TBST) rock for 10 min to wash

Addition of 2o Antibody•Discard wash solution

•Pour secondary antibody onto the membrane and gently

rock for 30 minutes

•Secondary antibody will bind to the primary antibody

•Quickly rinse membrane in 50ml of wash buffer and discard

the wash buffer

•Add 50ml of wash leave for 3 minutes on the rocking

platform

Production of 2o Antibody

• Antibodies from the first animal model are injected into a

second animal model

• The second animal produces antibodies against the first

antibody (secondary antibody)

• The secondary antibody is purified and conjugated to a

colorimetric substrate or to an enzyme that can cleave a

colorimetric compound

Detection of Protein•Discard wash solution

•Add the enzyme substrate (HRP color detection reagent) to

the membrane

•Incubate for 10 minutes

•The colorimetric substrate is cleaved by the enzyme

conjugated (attached) to the secondary antibody

Other detection methods

•Chemiluminescent substrates

• Film or cooled CCD camera

•Infrared emitting side groups

• Special machine

•Quantum dots

• Epi UV and CCD camera with EtBR filter

Western vs ELISAEnzyme-Linked Immunosorbant Assay

ELISA

- Quick results

- Primary screening

- Identifies proteins by

antibody specificity only

- With correct controls

can be quantitative

Western Blot

- Confirm ELISA results

- More specific

- Identifies proteins by both antibody specificity and size

-Difficult to use for quantification

Questions ?