Protein Detection Methods & Western

Blots

1. Sodium Dodecyl Slufate (SDS)-Polyacrylamide Gel

Electrophoresis (SDS-PAGE)

• Visualize many proteins at once• 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.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•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

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•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

Gly

proteinsCl-

Resolving gel: range 5-20%

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• 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 staindiffer 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

Stain

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 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 (?)

According to Bio-Rad

• Tangible results

• Link to careers and industry (?)

• Standards-based (?)

Why use Western blotting?

•Can specifically determine if your protein of

interest is in present in a crude protein

preparation

Transfer

Proteins from

the gel to the

nitrocellulose

membrane

60 minutes

200 mAElectric Current

Protein Transfer

200 mA

Blotting buffer

1x Tris glycine

with 20%

methanol,

0.1% SDS

Electric Current

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 non-

specifically to the membrane

Blocking membrane before addition of antibody

specifically 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

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 10ml of the enzyme substrate (HRP color

detection reagent) onto the membrane

•Incubate for 10 minutes

•The colorimetric substrate is cleaved by the

enzyme conjugated (attached) to the

secondary antibody

Experiment 4

Run two different SDS-PAGE gels:

1. detect proteins by rapid Coomassie staining

2. detect protein by Western blot

Questions