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Protocols
book

2013/2014

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Contents

Protocols

SECTION 1: Antibodies and antibody structure . . . . . . . .3

SECTION 2: Formats of antibody and . . . . . . . . . . . . . . . .6
antibody purification

SECTION 3: Choosing an antibody and antibody dilution . . .7

SECTION 4: Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . .9
4.1 How single fluorochromes work . . . . . . . . . . . . . . . . . . . .9
4.2 Fluorochrome table (excitation and emission wavelengths) . .11

SECTION 5: Western blot . . . . . . . . . . . . . . . . . . . . . . . . .13
5.1 Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . .15

Lysis buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15
Protease and phosphatase inhibitors . . . . . . . . . . . . . . .16
Preparation of lysate from cell culture . . . . . . . . . . . . . . .16
Preparation of lysate from tissues . . . . . . . . . . . . . . . . . .16
Determination of protein concentration . . . . . . . . . . . . . .17
Preparation of samples for loading into gels . . . . . . . . . .17

5.2 Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18
Preparation of PAGE gels . . . . . . . . . . . . . . . . . . . . . . . .18
Positive controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19
Molecular weight markers . . . . . . . . . . . . . . . . . . . . . . . .19
Loading samples and running the gel . . . . . . . . . . . . . . .19
Use of loading controls . . . . . . . . . . . . . . . . . . . . . . . . . .20

5.3 Transfer of proteins and staining . . . . . . . . . . . . . . .20
Visualization of proteins in gels . . . . . . . . . . . . . . . . . . . .20
Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20
Visualization of proteins in membranes: Ponceau Red . . . . .22
Blocking the membrane . . . . . . . . . . . . . . . . . . . . . . . . . .22
Incubation with the primary antibody . . . . . . . . . . . . . . . .22
Incubation with the secondary antibody . . . . . . . . . . . . . .22
Development methods . . . . . . . . . . . . . . . . . . . . . . . . . . .23

5.4 Western blot references . . . . . . . . . . . . . . . . . . . . . . .23

5.5 Troubleshooting tips – Western blotting . . . . . . . . . .23

SECTION 6: Immunohistochemistry . . . . . . . . . . . . . . . .27
6.1 IHC-Paraffin embedded . . . . . . . . . . . . . . . . . . . . . . . . .27

Optimizing a new antibody for IHC-P . . . . . . . . . . . . . . .29
Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .29
Deparaffinization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .29
Antigen retrieval . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .29
Immunostaining and detection protocol . . . . . . . . . . . . .33
Resources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .36

6.2 IHC-Frozen sections (IHC-Fr) . . . . . . . . . . . . . . . . . . . .36
6.3 Immunocytochemistry (ICC) . . . . . . . . . . . . . . . . . . . . .37
6.4 IHC and ICC fixation and permeabilization tips . . . . . . .38
6.5 Perfusion fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .39
6.6 Mouse on mouse staining tips . . . . . . . . . . . . . . . . . . . .40
6.7 Troubleshooting tips – IHC/ICC . . . . . . . . . . . . . . . . . . .41

SECTION 7: ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .43
7.1 Indirect ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .43
7.2 Direct ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .45
7.3 Sandwich ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .46
7.4 Troubleshooting tips – ELISA . . . . . . . . . . . . . . . . . . . . .48

SECTION 8: Flow Cytometry . . . . . . . . . . . . . . . . . . . . . . .51
8.1 The Flow Cytometer: Fluidics . . . . . . . . . . . . . . . . . . .51
8.2 The Flow Cytometer: Measurement of forward
and side scatter of light . . . . . . . . . . . . . . . . . . . . . . . . . . . .52
8.3 The Flow Cytometer: Measurement of scattered
light and fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . .52
8.4 Antibody staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . .56
8.5 Selecting a fluorochrome conjugate . . . . . . . . . . . . . . .57

SECTION 9: Immunoprecipitation (IP) Protocol . . . . . . .58
9.1 Lysis buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .58
9.2 Preparation of lysates . . . . . . . . . . . . . . . . . . . . . . . . . .58
9.3 Pre-clearing the lysates . . . . . . . . . . . . . . . . . . . . . . . .59
9.4 Immunoprecipitation . . . . . . . . . . . . . . . . . . . . . . . . . . .60
9.5 Choosing the correct beads . . . . . . . . . . . . . . . . . . . . .61
9.6 Procedure for crosslinking the antibody to the beads . .61
9.7 Using IgM antibodies for immunoprecipitation . . . . . . .63
9.8 Protein L beads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .63
9.9 Troubleshooting tips – IP . . . . . . . . . . . . . . . . . . . . . . . .64

SECTION 10: ChIP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .65
10.1 X-ChIP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .65
10.2 Troubleshooting tips – ChIP . . . . . . . . . . . . . . . . . . . .69

SECTION 11: Buffers and stock solutions . . . . . . . . . . .71
11.1 Standard PBS, TBS, TBS Tween . . . . . . . . . . . . . . . . .71
11.2 Western blot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .71
11.3 IHC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .73
11.4 ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .74
11.5 Flow Cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .74
11.6 IP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .74
11.7 ChIP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .74

SECTION 12: Antibody storage guide . . . . . . . . . . . . . .75

Welcome to Abcam

About us . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
More than just antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Comprehensive information . . . . . . . . . . . . . . . . . . . . . . . . . .1
100% support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2

Fast global delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2
Abreviews® . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2
Sign up for an Abcam account . . . . . . . . . . . . . . . . . . . . . . . .2

Technical help

Online resources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .76
Datasheet guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .76
Online protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .77

Online poster library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .77
Abcam blog . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .77

Ordering and contact details

Contact details . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .78 Local offices and distributors . . . . . . . . . . . . . . . . . . . . . . . .79

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Welcome to Abcam

About us

Abcam is a provider of protein research tools and
services, with an unrivalled range of products and expert
technical support. Abcam is committed to providing
scientists with an extensive choice of reagents and tools,
with the most comprehensive, honest and up-to-date
datasheets and customer reviews, fast delivery and
helpful customer service & technical support.

More than just antibodies

Hundreds of new products are added to our catalog each
month including a growing range of non-antibody products:

• Proteins and peptides
• Biological dyes and thermo-reversible gels
• Cell viability, cell proliferation and senescence kits
• EasyLink antibody conjugation kits
• ELISA, ELIPAIR, ELISPOT and FLUOROSPOT kits
• Epigenetic kits
• IHC kits and reagents
• Western blot reagents
• Biochemicals

Comprehensive information

Our products are accompanied by a wealth of technical data:

• Comprehensive datasheets
• Customer reviews (Abreviews®)
• Protocols and troubleshooting tips
• Frequently asked questions

We have a policy of honesty and so we share everything
that we know about our products, as soon as we know it
– it’s all on the datasheet.

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100% support

We’re committed to providing the highest levels of support
to our customers, which is why our technical teams are
scientists from a wide range of research backgrounds,
experienced in protein detection.

As part of our Abpromise, we guarantee high product
quality and expert support including:

• A refund or replacement if the product doesn’t work as
stated on the datasheet
• Support in six languages and a response to your inquiry
within 24 hours
• Extensive multi-media resources – pathways, posters,
technical webinars and more…

Fast global delivery

We currently ship to 140 countries. To get our products to you as quickly as possible we have extended our cut off
times for same day despatch of in-stock orders. Customers in North America can now place orders up to 8.00pm
EST, and in mainland Europe up to 4.00pm GMT, for next day delivery in most countries.

Abreviews® – independent customer reviews

Our Abreviews system allows customers to feedback
about the performance of our products. We share this
information with all of our customers on our datasheets,
whether the review is positive or negative.

This up-to-date information provides useful data about new
applications, optimal dilution conditions and images of our
products at work.

For further information visit: www.abcam.com/Abreviews

Sign up for an Abcam account!

Take full advantage of the numerous benefits of opening an Abcam account:

• Get direct access to expert scientific support
• Buy quickly and efficiently online
• Create and submit Abreviews
• Receive information about special offers, events and news
• View all orders, inquiries and conference registrations

To register visit: www.abcam.com/register

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SECTION 1: Antibodies and antibody structure
Antibodies, also called immunoglobulins (Ig), are glycoproteins that are capable of specifically binding to antigens that caused their production
in a susceptible animal. They are produced in response to the invasion of foreign molecules in the body. Antibodies exist as one or more
copies of a Y-shaped unit, composed of four polypeptide chains. Each Y contains two copies of a heavy chain, and two copies of a light chain,
named as such by their relative molecular weights. The top of the Y shape contains the variable region, which is the antigen binding site.

The light chains of any antibody can be classified as either a kappa (�) or lambda (�) type (based on small polypeptide structural differences);
however, the heavy chain determines the class, or isotype, of each antibody.

Antibody structure:

Heavy chains
There are five types of mammalian Ig heavy chains denoted by the Greek letters: alpha (�), delta (�), epsilon (�), gamma (�), and mu (�).
These chains are found in IgA, IgD, IgE, IgG, and IgM antibodies, respectively. Distinct heavy chains differ in size and composition; � and �
contain approximately 450 amino acids, while � and � have approximately 550 amino acids.

Each heavy chain has two regions, the constant region and the variable region. The constant region is identical in all antibodies of the same
isotype, but differs in antibodies of different isotypes. Heavy chains �, � and � have a constant region composed of three tandem (in a line)
Ig domains, and a hinge region for added flexibility; heavy chains � and � have a constant region composed of four Ig domains. The variable
region of the heavy chain differs in antibodies produced by different B cells, but is the same for all antibodies produced by a single B cell or
B cell clone. The variable region of each heavy chain is approximately 110 amino acids long and is composed of a single Ig domain.

Light chains
In mammals there are only two types of light chains, which are called lambda (�) and kappa (�). A light chain has two successive domains:
one constant domain and one variable domain. The approximate length of a light chain is 211 to 217 amino acids. Each antibody contains two
light chains that are always identical; only one type of light chain, � or �, is present per antibody in mammals. Other types of light chains,
such as the iota (�) chain, are found in lower vertebrates like Chondrichthyes (cartilaginous fishes) and Teleostei (ray-finned fishes).

S SS S
C

H
3

C
H

2

C
H 1C

L

V
H

V
L

S
S

C

antigen
binding

biological
activity

Fc Region

(Fab’)
2

Fab

N

C C

N
N N

C

CHO

C
H

3
C

H
2

C
H

1

C
L

V
H

V
L

S
S

CHO

light chain k or l

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Fab and Fc regions
Direct-conjugated antibodies are labeled with an enzyme or fluorochrome in the Fc region. The Fc region also anchors the antibody to the
plate in ELISA procedures and is also recognized by secondary antibodies in immunoprecipitation, immunoblots and immunohistochemistry.
Antibodies can be cleaved into two F(ab) and one Fc fragments by the proteolytic enzyme papain, or into just two parts: one F(ab)2 and one
Fc at the hinge region by the proteolytic enzyme pepsin. Fragmenting IgG antibodies is sometimes useful because F(ab) fragments (1) will
not precipitate the antigen; and (2) will not be bound by immune cells in live studies because of the lack of an Fc region. Often, because of
their smaller size and lack of crosslinking (due to loss of the Fc region), Fab fragments are radiolabeled for use in functional studies.
Interestingly, the Fc fragments are often used as blocking agents in histochemical staining.

Antibody isotypes:

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In mammals, antibodies can be divided into five isotypes or classes: IgG, IgM, IgA, IgD and IgE, based on the number of Y units and the type
of heavy chain. Heavy chains of IgG, IgM, IgA, IgD, and IgE, are known as gamma (�), mu (�), alpha (�), delta (�), and epsilon (�),
respectively. The isotypes differ in their biological properties, functional locations and ability to deal with different antigens, as depicted in the
table below:

Chicken IgY
There are several advantages to choosing chickens, rather than rabbits or goats to produce polyclonal antibodies.

1. Chickens are not mammals and therefore are more able to make high-avidity antibodies to mammalian antigens (especially highly conserved
mammalian proteins).

2. To our knowledge, it is the most ethical way to produce polyclonal antibodies. There is no need to bleed the chickens; simply collect the
eggs. Our hens are kept in flocks of six in coops that have outside runs and our unique egg identification system allows our chickens to live
freely.

3. A single chicken can produce an enormous amount of antibodies, up to 3 g of IgY per month, which is 10-20 times the amount of a rabbit.
Furthermore, compared to rabbits, chickens produce antibodies more quickly. High-titer antibodies are available from eggs as early as day
25.

4. By having the IgY packaged conveniently in eggs, you can collect and store eggs over a long period of time and retroactively purify the IgY
from the eggs of desired titer/avidity.

5. It is cheaper to feed and house chickens than rabbits.

6. IgY is a stable antibody sharing the following characteristics with mammalian IgG:
• Divalent
• Degraded by papain to yield divalent Fab fragment
• May be enzyme-labeled, biotinylated and gold–labeled by standard procedures

7. Fc region of chicken IgY is sufficiently different from mammalian IgG:
• Reduces background by not binding to mammalian rheumatoid factors or other naturally occurring anti-mammalian antibodies (e.g. HAMA)
• Does not activate mammalian complement systems
• Does not bind to mammalian Fc receptors
• Does not bind to Staphylococcal protein A or protein G

Class/
subclass

Heavy chain
Light
chain

Molecular
weight (kDa)

Structure Function

IgA1
IgA2

�1
�2

� or � 150 to 600
Monomer to

tetramer

Most produced Ig. Found in mucosal areas, such as the
gut, respiratory and urogenital tract, and prevents their
colonization by pathogens. Resistant to digestion and is
secreted in milk.

IgD
� � or � 150 Monomer

Function unclear; Works with IgM in B-cell development;
mostly B-cell bound.

IgE
� � or � 190 Monomer

Binds to allergens and triggers histamine release from
mast cells, and is involved in allergy. Also protects
against parasitic worms.

IgG1
IgG2a
IgG2b
IgG3
IgG4

�1
�2
�3
�4

� or � 150 Monomer
Major Ig in serum. Provides the majority of
antibody-based immunity against invading pathogens.
Moderate complement fixer (IgG3); can cross placenta.

IgM
µ � or � 900 Pentamer

First response antibody. Expressed on the surface of B
cells and in a secreted form with very high avidity.
Eliminates pathogens in the early stages of B
cell-mediated immunity before there is sufficient IgG.

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SECTION 2: Formats of antibody and antibody purification
Centrifugation and filtration are standard laboratory techniques for sample clarification of serum, ascetic fluid and tissue culture supernatant.
These techniques remove lipid and particle matter which can block chromatographic columns. For some materials buffer exchange and
desalting may also be necessary. Ammonium sulphate precipitation is a further preparation step often used with ascetic fluid to concentrate
the immunoglobulins.

Antiserum
Polyclonal antibodies are often available in relatively unpurified formats, described as “serum” or “antiserum”. Antiserum refers to the blood
from an immunized host from which clotting proteins and red blood cells have been removed. The antiserum will contain
antibodies/immunoglobulins of all classes as well as other serum proteins. In addition to antibodies that recognize the target antigen, the
antiserum also contains antibodies to various non-target antigens that can sometimes react non-specifically in immunological assays. For
this reason, raw antiserum is often purified to eliminate serum proteins and to enrich the fraction of immunoglobulin that specifically reacts
with the target antigen.

Tissue culture supernatant
Monoclonal antibodies may be grown as hybridoma cell cultures (cells secreting antibodies) and harvested as hybridoma tissue culture
supernatants.

Ascites fluid
Monoclonal antibodies can be produced by growing hybridoma cells within the peritoneal cavity of a mouse (or rat). When injected into a
mouse, the hybridoma cells multiply and produce fluid (ascites) in its abdomen. This fluid contains a high concentration of antibody which can
be harvested, providing higher antibody yields than hybridoma cell-culture.

Antibody purification
Polyclonal antiserum or monoclonal ascites fluid / tissue culture supernatant is commonly purified by one of three methods:

1. Protein A/G purification
Protein A/G purification makes use of the high affinity of Staphylococcus aureus protein A or Streptococcus protein G to the immunoglobulin
Fc domain. Protein A/G purification eliminates serum proteins from raw antiserum, but it does not eliminate the non-specific immunoglobulin
fraction. Consequently, protein A/G purified antiserum may still possess a small amount of undesirable cross reactivity.

2. Affinity purification
Affinity purification isolates a specific protein or group of proteins with similar characteristics. The technique separates proteins on the basis
of a reversible interaction between the proteins and a specific ligand coupled to a chromatographic matrix.
Antigen affinity purification takes advantage of the affinity of the specific immunoglobulin fraction for the immunizing antigen against which it
was generated. Antigen affinity purification results in the elimination of the bulk of the non-specific immunoglobulin fraction, while enriching
the fraction of immunoglobulin that specifically reacts with the target antigen. The resulting affinity purified immunoglobulin will contain primarily
the immunoglobulin of desired specificity.

3. Pre-adsorption
Polyclonal antibodies are sometimes pre-adsorbed. This means they have been adsorbed with other proteins, or serum from various species,
to eliminate any antibody that may cross-react. The resulting purified antibody should be very pure and specific and any cross-reactivity
should be significantly reduced.

Antibody purification at Abcam

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SECTION 3: Choosing an antibody and antibody dilution
There is often more than one antibody available for any given target. To narrow the range of choice, several aspects of the experiment need
to be considered:

1. Type of assay or application
2. Nature of the sample
3. Species of the sample
4. Species of the antibody host
5. Labeling and detection of the antibody

The Abcam website has a useful search function. Entering the name of the protein or other target in the search box will generate a list that
can be filtered by product type, target, applications tested, species reactivity, host species, clonality, and conjugation.

Application
Antibody datasheets list the applications that have been tested and found to work. If an application is not listed, this does not mean that the
antibody is not suitable. It simply means that it has not been tested and it is unknown how the antibody will perform. When an application has
been tested and found not to work, this will be noted on the datasheet.

Nature of the sample
The nature of the sample will dictate which antibody will work best. At least two aspects need to be considered:

1. The region of the protein you wish to detect. Antibodies are generated by immunization of host animals with a variety of immunogenic
substances including full-length proteins, protein fragments, peptides, whole organisms (for example bacteria), or cells. The immunogen is
generally described on the datasheet (though in many cases an exact description of the immunogen is not available for proprietary reasons).
If trying to detect a protein fragment or a specific isoform or region of the full-length protein, one needs to be sure to choose an antibody that
is raised against an immunogen that is identical to or contained within the fragment or region. If trying to detect a cell surface protein on live
cells by FACS, you need to choose an antibody that is raised against an extracellular domain of the protein.

2. Processing of the sample. Some antibodies require samples to be processed or treated in a specific manner. For instance, many antibodies
will only recognize proteins that have been reduced and denatured, presumably because this reveals epitopes that would otherwise be
obscured by secondary and tertiary folding of the proteins. On the other hand, some antibodies will only recognize epitopes on proteins in
their native, folded state. (For Abcam antibodies used for western blotting, samples should be reduced and denatured unless otherwise noted
on the datasheet). When searching for an antibody for immunohistochemistry, it should be noted that some antibodies are only appropriate
for unfixed frozen tissue. For others, an antigen retrieval step that reverses the cross-links introduced by formalin fixation is necessary because
they are incapable of binding to their targets in formalin-fixed, paraffin-embedded tissues. These restrictions on use will be noted in the
applications section of datasheets.

Species of sample
The chosen antibody should have been raised against the same species you are studying, although the antibody may react with the same
target protein from other species sharing sufficient amino acid sequence homology. If the sample is not from one of the species listed, this
does not mean that the antibody will not detect the protein, but rather that the species has not been tested and we are reluctant to comment
on its suitability. A prediction of cross-reactivity can be made based on sequence similarity: Abcam is now proud to have ExPASy and NCBI
BLAST links on the datasheets to compare amino acid sequence homology among different species.

Choosing the species of primary antibody host
In general, the species of the host animal in which an antibody was raised is important when using a conjugated secondary antibody to detect
an unconjugated primary. For immunohistochemistry, the primary antibody should be raised in a species as phylogenetically different as
possible from the species of the sample. This is to avoid potential cross-reactivity of the secondary anti-immunoglobulin antibody with
endogenous immunoglobulins in the sample. For instance, a primary antibody used to detect a protein in a sample from a mouse should not
be raised in mouse or rat. A primary antibody raised in rabbit will be a more appropriate choice, followed by an anti-rabbit IgG secondary
antibody conjugated to a detection molecule (enzyme, fluorochrome, biotin, etc.). This issue can be avoided if a conjugated primary antibody
is available. For other techniques using samples that do not contain …