5/8/2020

ImmunoBLOTTING

Ankur Gautam, PhD.,

Merck High End Skill Development Centre
Chandigarh, India
27th April, 2020

Contents
❑ Blotting and types

❑ Western blotting

❑ Sample preparation

❑ SDS-PAGE

❑ Transfer

❑ Blocking

❑ Development

❑ Detection analysis

❑ Application

Blotting
Method of transferring biomolecules from gel to membrane

Northern Western
Southern (RNA) (Protein)
(DNA)

Dr. Harry Towbin

Dr. W. Neal Burnette
Prof. Edvin Southern Dr. George Stark 1979-1981
1975 1977

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Western Blotting
Identification and detection of specific protein in a sample (lysate)

❑ Uses an electrical current to transfer sorted proteins from gel to

Nitrocellulose membrane
-More stable than a thin Polyacrylamide gel
❑ Easier to stain and record data
-Longer lived, can last months-years if stored properly.

❑ Also known as “Immunoblotting”

❑ World’s most widely used biochemical method.

❑ Rapid and sensitive

SDS-PAGE
Sample Transfer
Electrophoresis
preparation

Immunoblotting
Blocking

Analysis Detection Development

Sample Preparation

Extraction
Proteins

Lysis Buffer

Cell
Loading buffer
(Laemmli Buffer)

Sample ready
for electrophoresis
Heat at 90-100°C
5 min

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Loading buffer
Reduce disulfide ridges
between cysteines Tracking dye
Migrate with the leading edge
DTT
BPB of the proteins being separated
Denatures, linearizes ß-ME
the proteins,
coating them in Increases density of sample
negative charge Allow the samples to sink
SDS Glycerol into each well
Loading
buffer

Sodium Dodecyl Sulfate

Polyacrylamide Gel Electrophoresis

SDS-Polyacrylamide Gel
Separation of proteins from a sample (lysate)

Components
Reagent Function
Acrylamide It can forms linear polymers
Bis-acrylamide create crosslinks between acrylamide

Tris Hcl Buffer (7-9 pH range)

10% SDS Denature the protein

10% APS oxidizing agent (produce free radicals)
TEMED Catalyze the polymerization
β-ME Reduces the di-sulfide bonds

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SDS-Polyacrylamide Gel Preparation

Separating Gel (10-15%)
• Acrylamide : Bisacrylamide solution
• Water Stacking Gel
• Tris-HCl, pH 8.8 • Dilute
• 10%SDS • 3-5%
• 1% APS • Larger pore size
• TEMED (add in the last) • pH 6.8

Stacking Gel (3-5%) Separating gel

• Acrylamide : Bisacrylamide solution • Concentrate
• Water • 10-15%
• Tris-HCl, pH 6.8 • small pore size
• 10%SDS • pH 8.8
• 1% APS
• TEMED (add in the last)

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SDS-PAGE Electrophoresis

_
Protein first
Running Buffer
Stack and
• Tris-Base, pH 8.3
• Glycine
then
• SDS
Separated
on the basis of
their size or
Mol Weight

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Q1. Which of the following technique was discovered first?

(A) Southern blotting

(B) Northern blotting
(C) Western blotting

Correct Answer: A

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Q2. DTT is used for

(A) Breaking non-covalent bonds

(B) Breaking disulfide bonds
(C) Breaking ionic bonds
(D) Breaking Van der waal bonds

Correct Answer: B

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Q3. In SDS-PAGE, separation of proteins are based on

(A) Charge
(B) Shape
(C) Size
(D) Both charge and size

Correct Answer: C

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Transfer of Protein

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Orientation of Membrane & Gel is Important

Filter paper
Gel
Fiber pad
Membrane

Filter paper
Fiber pad

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Preparation of transfer stack

Cassette

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Power
Setting up Supply

Transfer + _
Transfer Buffer
• Tris-Base
• Glycine
• Methanol

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Transfer of Proteins to Membrane

+
-

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Q4. In Transfer, Gel and membrane should be placed at which side?

(A) Gel at negative and membrane at positive electrode

(B) Gel at positive and membrane at negative electrode
(C) Both at positive electrode
(D) Both at negative electrode

Correct Answer: A

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Q5. What are the chemicals needed for making transfer buffer?

(A) Tris, glycine and ethanol

(B) Tris, glycine and methanol
(C) Tris, glycine and acetone
(D) Tris, glycerol and methanol

Correct Answer: B

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Blocking of Blot

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Blocking
➢ Prevent non-specific binding of the antibodies,
➢ Blocking of unbound membrane sites (BSA, low fat Milk or skimmed milk, etc)
➢ Leads to low backgrounds.

Before blocking After blocking

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Blot Development

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Requirements

❑ Blot with transferred protein

❑ Wash solution (TBST)

❑ Primary antibody

❑ Secondary antibody conjugated with enzyme

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Antibody Types
Primary Antibody Secondary Antibody

Anti-Mouse
Mouse anti-human
Anti-rabbit
Rat anti-mouse
Anti-goat
Rabbit anti-human

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Generation of Primary & secondary Antibody

Primary Ab

Rabbit

Human Protein
(Antigen)

Primary Ab
(Mouse anti-human)
Secondary Ab
(anti-mouse)

Mouse

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Blot Development

Blocked membrane

Washings
Primary Abs

Washings Washings
Detection

Secondary Abs

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Protein Detection

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Protein Detection
➢ Based on enzyme-linked reaction,

➢ Utilizing secondary antibodies covalently bound to enzymes

(Horseradish Peroxidase, Alkaline phosphatase)

➢ Conjugated enzyme catalyzes the degradation of specific substrates, resulting

in signal generation.
❑ Chromogenic - Colored product

❑ Chemiluminescent -Light

❑ Fluorescent - Fluorescence

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Colorimetric detection
When the chromogenic substrate is added, the enzyme catalyses a reaction and produces a colored
precipitate that is deposited on the blot. This precipitate is easily visible and can be photographed

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Chemiluminescence Detection
Chemiluminescence is a process by which chemical reaction involving a substance results in the
release of energy in the form of light.

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Fluorescence detection
The dyes fluoresce at a particular wavelength and can be detected by imaging the blot.

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Q6. Blocking is carried out for

(A) To increase the binding of Abs to membrane

(B) To prevent nonspecific binding of Abs to membrane
(C) For detection of proteins
(D) For blot development

Correct Answer: B

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Q7. In Chemiluminescence, detection of proteins is based on

(A) Colored product

(B) Fluorescence
(C) Light
(D) Infra red rays

Correct Answer: C

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Q8. The dark background in the blot could be due to

(A) Insufficient blocking
(B) Too less protein
(C) Too less antibody
(D) Too much protein

Correct Answer: A

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SNAP i.d.® 2.0

The latest Technology

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SNAP i.d.® 2.0 Protein Detection System

Rapid system takes protein detection to new dimensions

Sample SDS-
Transfer Blocking Development Detection
preparation PAGE

30 min 60 min 2-3 hrs 1-12 hrs 5-15 hrs 15 min

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Applications

❑ Is my protein expressed? ❑ Do the levels change in response to

treatment?

1 2 3 1 2 3

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THANKS

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