antibody labeling and detection · 2013-12-05 · antibody conjugation kits can be used and where...

Discover more at abcam.com/Conjugation

Antibody Labeling and Detection Tips for Success using Antibody Conjugation Kits

Discover more at abcam.com/Conjugation 1

Contents

Introduction and applications of directly labeled primary antibodies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2

Benefits of direct assays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3

Labeling chemistry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4

Conjugation Kits product list. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5

Tips for selecting the right type of conjugate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Enzymatic and biotin detection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Fluorescent detection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8

Tips for using tandem dyes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Tips for successful antibody labeling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10

Antibody purification and concentration kits. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11

Publications using conjugation kits. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Discover more at abcam.com/Conjugation2

Introduction and applications of directly labeled primary antibodies

Antibodies are a valuable tool in immunoassays to detect and quantify antigens due to their specificity and affinity for an antigen. Immunochemical assays typically rely on a ‘primary’ antibody that binds to a specific antigen and a label that is incorporated into the assay in order to provide measurability. This label is incorporated into the assay using one of two methods – indirect or direct detection methods. Indirect detection relies on a labeled secondary reagent, whereas direct detection relies on a labeled primary antibody the benefits of which are outlined in the section.

There are a wide range of commonly used labels for immunochemical detection techniques. Some of these techniques and the labels used are shown in Table 1. Directly labeled primary antibodies can be used in most if not all of these techniques.

Table 1. Techniques and corresponding labels that directly labeled primary antibodies can be used in.

Abcam antibody conjugation kits can be used for the direct conjugation of a chosen label to primary antibodies and other proteins.

The publications on page 13 demonstrate some of the applications in which antibodies labeled with Abcam antibody conjugation kits can be used and where researchers have benefited from direct assays.

Immunoassay Labels

Western blotting Enzymes (e.g. HRP or AP), Fluorescent dyes

ELISA Enzymes, Biotin/Streptavidin, Fluorescent dyes

Immunocytochemistry (ICC) Fluorescent dyes

Immunohistochemistry (IHC) Enzymes, Biotin/Streptavidin, Fluorescent dyes, Gold

Flow Cytometry Fluorescent dyes, Tandem dyes

Electron Microscopy Gold

Immunoprecipitation Protein A/G, Magnetic beads

Raman Spectroscopy Gold


Benefits of direct assays

Typically performing a direct assay can be difficult as finding commercially available directly labeled primary antibodies for an antigen being studied can be a challenge. This can be solved by using a secondary antibody – however using a secondary antibody can present a number of other challenges that can be solved by using a directly labeled primary antibody in a direct assay.

The advantages and disadvantages of indirect vs. direct assays are outlined in Table 2 below.

Table 2. Advantages and disadvantages of direct vs. indirect labeling methods.

Method Advantages Disadvantages

Indirect Allows signal amplification Non-specific background may result from the secondary antibody cross-reactivity

Immunoreactivity of the primary antibody is not affected by traditional labeling methods

Extra incubation steps are required

The ratio of secondary to primary antibody has to be optimized

Time consuming protocol

Wash steps may remove bound primary antibody and increased sensitivity may not be realised

Direct Quick methodology since only one antibody is used

Immunoreactivity of the primary antibody may be reduced through traditional labeling methods

No need for secondary antibodies Little signal amplification

No cross-reactivity of secondary antibody

Same primary antibody host species can be used with same sample species (e.g. mouse-on-mouse)

Different fluorescent dyes can be used in the same experiment – thus, multi-plex assays are easier to run


Labeling chemistry

Antibodies are made of the same building blocks as all proteins – amino acids including the side chain of a lysine that terminates in a primary amine (-NH2). There are four main methods by which an antibody can be chemically labeled:

1. NHS esters – activated fluorescent dyes (with inbuilt NHS ester/succinimidyl ester) react with antibodies. Excess dye is then removed using methods such as column chromatography. 2. Heterobifunctional reagents – for labels that are protein molecules (e.g. HRP, AP or R-PE). Both antibody and label contain multiple amines which can be modified so that lysines on one molecule (e.g. antibody) form a reactive group (X) and using a heterobifunctional reagent, a reactive group (Y) is formed from lysines on the label. X and Y groups can then react forming a heterodimeric conjugate. 3. Carbodiimides – these types of reagents (e.g. EDC) create covalent links between an amine- (i.e. provided by lysine) and carboxyl- containing molecules. Carodiimides are not typically used to attach dyes or protein labels to antibodies, but can be used to attach carboxylated particles such as latex particles or magnetic beads. 4. Sodium periodate – this chemical is not typically used to attach most labels, however it is important for the conjugation of HRP. Periodate can activate carbohydrate chains on HRP molecules creating aldeyhyde groups which can react with antibody molecules (Note: not always necessary as HRP itself also contains lysines).

Abcam antibody conjugation kits simplify the labeling process through eliminating column separation steps and the associated issues caused by this process:

• Loss of material• Sample dilution during column chromatography• Batch to batch variation• Challenges in scaling up

The labeling process for Abcam antibody conjugation kits is shown in the figure below, and although the protocol is simple, it is an intricate and refined chemical process ensuring conjugates are produced in a gentle and controlled manner.

Fig. 1 Description of the rapid labeling procedure with Abcam antibody conjugation kits.


Conjugation kits product list

Abcam antibody conjugation kits can be used in Western Blotting, ELISA, Immunohistochemistry (IHC), FRET Immunofluorescence, FACS analysis and other immunoassay based techniques.

Four sizes are available dependent on the conjugate:

• 3x10μg - allows 3 reactions, each up to 30μg of antibody• 1x100μg - allows 1 reaction, up to 100μg of antibody• 3x100μg - allows 3 reactions, each up to 300μg of antibody• 1x1mg - allows 1 reaction, up to 1mg of antibody

GOLD conjugates

Conjugates for enzymatic detection

Conjugates for Biotin/Streptavidin detection

Conjugate Description/size Product code

GOLD Conjugation Kit (40nm, 20 OD) 30µg - 120µg ab154873

GOLD Conjugation Kit (80nm, 20 OD) 30µg - 120µg ab154876

GOLD-Biotin Conjugation Reagent (40nm, 10 OD) 200µl & 1ml ab154874

GOLD-Streptavidin Conjugation Reagent (40nm 10 OD) 200µl & 1ml ab154875

Conjugate Product code 3x10μg 1x100μg 3x100μg 1x1mg

AP ab102850

Glucose oxidase (GOx) ab102887

HRP ab102890

Conjugate Product code 3x10μg 1x100μg 3x100μg 1x1mg

Avidin ab102862

Biotin (type A) ab102865

Biotin (type B) ab102867

Streptavidin ab102921


Conjugates for fluorescent detection

Conjugate Product code Ex/Emmax 3x10μg 1x100μg 3x100μg 1x1mg

AMCA ab102847 350/445FITC ab102884 495/519Cy3® ab102874 548/561Rhodamine ab102915 570/590B-PE ab102871 546/575R-PE ab102918 535/575Texas Red® ab102924 595/613Cy5® ab102877 647/665Pe/Cy5® ab102893 565/666PerCP ab102907 447/678PerCP/Cy5.5® ab102911 477/694PE/Cy5.5® ab102899 565/693APC/Cy5.5® ab102855 650/694Cy5.5® ab102878 675/694APC/Cy7® ab102859 650/774PE/Cy7® ab102903 565/778Cy7® ab102882 753/775


Tips for selecting the right type of conjugate

The best conjugate to select for your experiments is application dependent. Some guidelines are given below or please contact our technical team for support in selecting the best conjugate for your experiments.

Enzymatic and biotin detectionFor enzymatic and biotin detection (e.g. in WB, IHC or ELISA), we suggest using a primary antibody directly conjugated to horseradish peroxidise (HRP), alkaline phosphatise (AP), or biotin. Both avidin and streptavidin bind very strongly to biotin, thus when using a biotinylated primary antibody for detection, avidin or streptavidin conjugated to an enzyme or fluorochrome bind onto multiple sites on the biotinylated primary antibody providing signal amplification and increased sensitivity. Glucose Oxidase (GO) is useful for developing multiplex enzymatic immunoassays; however it has the drawback of reduced sensitivity thus limiting its application.

The guide in Table 3 can be used to help with selecting the right enzymatic detection method for your experiment.


Enzyme Substrate Applications Advantages Drawbacks

HRP Chromogenic, soluble (TMB, ABTS, OPD...)

ELISA Easy to use Light sensitive coloration

Chromogenic, precipitating (CN, AEC, DAB,...)

WB, SB, IHC Easy to use Background in blood samples and some other tissues

Staining stability lower than AP

Fluorogenic (ADHP/resorufin)

Chemiluminescent

ELISA High sensitivity Need a fluorescent equipment

Luminol WB, SB, IHC High sensitivity Need radiographic equipment or light scanner

AP Chromogenic, soluble (pNPP)

ELISA Linear kineticOften more sensitive than HRP

Unstable

Chromogenic, precipitating (BCIP/NBT,...)

WB, SB, IHC Staining stability higher than HRP

Interfere with nuclear counterstain

Fluorogenic (4-MUP) ELISA, IHC Sensitivity Need fluorescence equipment

Table 3. Guide to enzymatic detection

Fluorescent detectionIf a laser light is involved (e.g. in Flow cytometry, ICC/IF or IHC) we suggest fluorescent detection with a primary antibody directly conjugated to a fluorochrome.

Due to their novel electronic configurations, fluorochromes have unique and characteristic spectra for absorption (excitation) and emission. It is necessary to have the appropriate equipment to capture a fluorescent signal in the relevant application.

Fluorescent detection enables the easy development of multiplex experiments by being able to detect multiple antigens in the same experiment with high levels of sensitivity and selectivity and using antibodies from the same species if directly conjugated.

Email [email protected] to request the Guide to Fluorochromes poster to help you with selecting the right single or tandem dye fluorochrome for your experiments.


Tips for using tandem dyes

When using fluorescent labels the use of fluorescent tandem dyes offers the possibility of designing multi-color experiments, particularly in flow cytometry. Tandem dyes are composed of two fluorescent dyes that are in close enough proximity and orientation that energy can be transferred from a donor molecule (e.g. PE) to an acceptor molecule (e.g. Cy5®) through fluorescence resonance energy transfer (FRET). The resulting fluorophore that is produced has the excitation properties of the donor and emission properties of the acceptor.

Some tips for using tandem dyes are outlined below:

1. Label cells at 4°C. Tandem dyes with APC may be degraded by cell activity, thus lowering the temperature reduces cell metabolism and maintains stability of the tandem dye. 2. Avoid extended exposure to light and extreme variations in temperature. Tandem dyes can be photobleached by light and extreme temperature variations can denature the tandem dye. 3. Be cautious during the fixation and permeabilization steps - avoid overfixation by using tandem dyes that can be fixed for short periods (e.g. 30 mins) and avoid vortexing the fixation and permeabilization buffers. 4. Be careful of cross-excitation of the acceptor molecule in multi-laser experiments, particularly where another fluorophore with similar excitation to the acceptor molecule is being used (e.g. when using a red laser with both PE/Cy5® and APC). 5. Impurities may affect FRET efficiency, thus, high quality reagents should be used in the experiment.


Tips for successful antibody labeling

Commercially available antibodies often contain substances such as stabilizing proteins or low molecular weight additives (e.g. BSA, glycine, Tris, sodium azide) that can interfere with your labeling experiments. In addition, these antibodies are often only available at lower concentrations because their compatibility with labeling methods may not have been a key consideration when the antibody was initially purified and formulated. (Note: Custom generated RabMAb antibodies can be provided purified and formulated for improved compatibility with conjugation kits)

Compatible additives that can be present in protocols with Abcam antibody conjugation kits are listed below:

Compatible additives:• up to 20mM Tris• up to 0.1% BSA*• up to 0.1% gelatin• up to 0.1% sodium azide• PBS pH6.5-8.5• up to 50% glycerol• salts (e.g. NaCl)• chelators (e.g. EDTA)

• 0.15M sodium chloride• 50mM HEPES• 0.02M potassium phosphate• 0.001% Tween• Proclin 300• 5% Trehalose• sugars (e.g. sucrose)

*Up to 1% BSA will work but yield lower quality conjugates.

We recommend avoiding the use of nucleophiles such as amino acids (e.g. glycine), blockers (e.g. ethanolamine) and thiols (DTT, mercaptoethanol) that could interfere with the conjugation reaction.

Incompatible additives:• Tris above 20mM• Urea• 50mM Imidazole• Glycine

• Ethanolamine• DTT• Mercaptoethanol

For most labeling reactions, the antibody will need to be reasonably pure (e.g. >90%, preferably >95%) and at a concentration of 1-4mg/mL for optimal results (0.5mg/mL is the lowest we recommend you try). Please refer to the relevant conjuation kit datasheet or protocol for recommended antibody concentration.

Our antibody purification and concentration kits (page 11), can be used to ensure your antibody is in suitable conditions for conjugation.

Find more tips for successful labeling at abcam.com/ConjugationFAQs.


Antibody purification and concentration kits

Many commercially available antibodies are often in a suitable form and concentration for labeling. However, there are situations where this is not the case. Antibody concentration and purification kits from Abcam can be used to optimize antibody solutions for conjugation. The figures below can be used to identify the most suitable kit to prepare your antibody.

Fig. 2 Guidelines for selecting the appropriate concentration and purification kit

Fig. 3 Suitability of the purification kits for each antibody host species

Species Protein A Antibody Purification Kit (ab102784)

Protein G Antibody Purification Kit (ab128747)

Mouse Antibody Purification Kit (ab128745)

Rabbit

Sheep

Mouse

Rat

Goat

/ Represents approximately 20% recovery* yellow most optimal

Approximate antibody recovery from different species using antibody purification kits


Publications using conjugation kits

Label: Cy3® Application: FRET Effects of pH on molecular mechanisms of chitosane-integrin interactions and resulting tight-junction disruptionsHsu LW, et al, Biomaterials, Jan 2013, 34(3):784-793, PMID: 23103155

Label: R-PE Application: Immunocytochemistry and In-vivo imaging*Intraoperative Imaging of Metastatic Lymph Nodes Using a Fluorophore-conjugated Antibody in a HER2/neu-expressing Orthotopic Breast Cancer Mouse ModelWu J et al, Anticancer Res, Feb 2013, 33(2):419-24, PMID: 23393332*Note: No product warranty for this application

Label: APC/Cy7® Application: Flow cytometryAPR-246/PRIMA-1MET rescues epidermal differentiation in skin keratinocytes derived from EEC syndrome patients with p63 mutationsShen J, et al, Proc Natl Acad USA, Feb 2013, 110(6):2157-62, PMID: 23355676

Label: FITC Application: Flow cytometry and cellular assayConstitutive dimerization of glycoprotein VI (GPVI) in resting platelets is essential for binding to collagen and activation in flowing blood.Jung SM, et al, J Biol Chem, Aug 2012, 287(35):30000-13, PMID: 22773837

Label: FITC Application: Flow cytometryIL-33 promotes the migration and proliferation of circulating fibrocytes from patients with allergen-exacerbat-ed asthma.Bianchetti L, et al, Biochem Biophys Res Commun, Sep 2012, 426(1):116-21, PMID: 22921786 Label: Streptavidin Application: ELISARapid det ection of Mycobacterium tuberculosis biomarkers in a sandwich immunoassay format using a waveguide-based optical biosensor.Mukundan H, et al, Tuberculosis, Sep 2012, 92(5):407-16, PMID: 22710249 Label: R-PE Application: Flow cytometryMultiple activating and repressive cis-promoter regions regulate TNFSF15 expression in human primary mononuclear cells.Gonsky R, Deem RL, Targan SR, Cytokine, Jul 2013, 63(1):36-42, PMID: 23642711 View more publications online.

Have you used an Abcam antibody conjugation kit in a recent publication? Please email [email protected] to tell us.

Discover more at abcam.com/Conjugation

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antibody labeling and detection · 2013-12-05 · antibody conjugation kits can be used and where...

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