First Class Secondary Antibodies

Jackson ImmunoResearch

For over 37 years Jackson ImmunoResearch have specialised in the manufacture of secondary antibodies, conjugates and purified immunoglobulins. Their products are made by scientists for scientists and are considered by many to be the gold standard. With their state of the art production facility in rural Pennsylvania and there European hub in Cambridgeshire they serve Research Institutes and Universities throughout the world.

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New! Anti-Camelid Secondary Antibodies

At Jackson ImmunoResearch, our mission is to develop the widest range of high quality secondary antibodies for the life science community. To support the growing adoption of camelid-derived heavy chain variable domain (VHH) technologies in a multitude of applications, including next-generation immunotherapeutics, medical imaging, analytical and diagnostic assay development and research discovery, JIR offers three new secondary antibody specificities for detection and quantification of camelid antibodies.

Immunoglobulins Poster

Download our guide to Immunoglobulins Structure and Function

Multiple Labelling Poster

Download our guide to Multiple Labeling with Secondary Antibodies

Customer Images

Image courtesy of Dr. Ehler, King’s College London.
Mouse, Rat and Rabbit Primary antibodies differentially detected with cross-adsorbed secondary antibodies from Jackson ImmunoResearch: 3 cell types are identified in this image. Red only cells show fibroblasts (positive for Rat Anti-tubulin); Smooth muscle cells are positive for both tubulin (red) and Mouse Anti-desmin (blue). Cardiomyocytes are identified in this image by their exhibition of green stripes, positive for Rabbit Anti-sarcomeric alpha-actinin.

Image courtesy of David Robertson
Breakthrough, The Institute of Cancer Research London.
Cultured mouse mammery gland tumour cells 4T1 grown on glass coverslips. Formalin fixed cells have been stained with anti alpha tubulin to reveal microtubules. The alpha tubulin was visualised with Alexa Fluor® 488 Goat anti Mouse IgG₁. Nucleus counterstained with DAPI.

Images by Brian McAdams and William Kennedy, University of Minnesota.
Jejunum villi superficial epithelium labeled with Ulex-biotin Streptavidin-Cy5 (016-170-084 blue), gastric K cells labeled with goat anti-GIP + donkey anti-goat-Cy2(705-175-147 green) and nerve fibers labeled with mouse anti-tubulin + donkey anti-mouse-Cy3 (711-165-151 red)

Featured Products

Product CodeProduct Description
115-545-003Alexa Fluor® 488-AffiniPure Goat Anti-Mouse IgG (H+L)
115-035-003Peroxidase-AffiniPure Goat Anti-Mouse IgG (H+L)
115-545-166Alexa Fluor® 488-AffiniPure Goat Anti-Mouse IgG (H+L) (min X Hu,Bov,Hrs,Rb,Rat Sr Prot)
715-545-151Alexa Fluor® 488-AffiniPure Donkey Anti-Mouse IgG (H+L) (min X Bov,Ck,Gt,GP,Sy Hms,Hrs,Hu,Rb,Rat,Shp Sr Prot)
715-035-151Peroxidase-AffiniPure Donkey Anti-Mouse IgG (H+L) (min X Bov,Ck,Gt,GP,Sy Hms,Hrs,Hu,Rb,Rat,Shp Sr Prot)
111-545-003Alexa Fluor® 488-AffiniPure Goat Anti-Rabbit IgG (H+L)
111-035-003Peroxidase-AffiniPure Goat Anti-Rabbit IgG (H+L)
111-545-144Alexa Fluor® 488-AffiniPure Goat Anti-Rabbit IgG (H+L) (min X Hu,Ms,Rat Sr Prot)
111-035-144Peroxidase-AffiniPure Goat Anti-Rabbit IgG (H+L) (min X Hu,Ms,Rat Sr Prot)
711-545-152Alexa Fluor® 488-AffiniPure Donkey Anti-Rabbit IgG (H+L) (min X Bov,Ck,Gt,GP,Sy Hms,Hrs,Hu,Ms,Rat,Shp Sr Prot)
711-035-152Peroxidase-AffiniPure Donkey Anti-Rabbit IgG (H+L) (min X Bov,Ck,Gt,GP,Sy Hms,Hrs,Hu,Ms,Rat,Shp Sr Prot)

FAQs

Manufacturing Questions

1. How do you determine protein concentration?

Jackson ImmunoResearch uses the Biuret assay to determine protein concentration.
If protein concentration is obtained by using the mass extinction coefficient of 1.4 for a 1 mg/ml solution at OD280, the result will be 10-15% lower than the concentration obtained by the Biuret assay.

2. What is the molecular weight of my product?

Jackson ImmunoResearch does not determine molecular weights by analytical methods. An approximate molecular weight of 160, 110 and 50 kDa can be used for whole IgG, F(ab’)2 and Fab fragment antibodies, respectively. To obtain an estimated molecular weight of any other product of interest, please contact Technical Service at help@jacksonimmuno.com.

3. Do you make custom antibodies?

For purchase of OEM products that have custom specifications (e.g. custom adsorptions, conjugations), please contact Technical Service at technical@stratech.co.uk. Jackson ImmunoResearch does not provide polyclonal or monoclonal antibody development or production services.

4. Are your products endotoxin-free?

Jackson ImmunoResearch does not assay products for endotoxin content. For applications requiring low endotoxin consider using an endotoxin removal column, available from Thermo Fisher, Charles River Laboratories, and other suppliers.

5. Why is there a discrepancy regarding the protein concentration, volume of dH2O used to reconstitute the lyophilized powder, and the nominal fill size reported on the spec sheet?

For products that are sold by weight (mg), Jackson ImmunoResearch fills vials with slightly more product that the nominal mg amount (“Size” on the spec sheet). To calculate the actual product amount in a vial, multiply the protein concentration indicated on the spec sheet by the volume of dH2O needed to rehydrate the lyophilized pellet.

General Application Questions

1. What are common causes of background?

Background may be caused by the primary antibody. Perform a control experiment excluding the primary antibody to isolate the secondary antibody as the cause of background.
Improper blocking of the tissues or cells.
Cross-reactivity of the labeled secondary antibodies with endogenous immunoglobulins on the tissues or cells.
Inadequate washing.
Reactivity of the labeled secondary antibody with immunoglobulins in the diluent.
Not diluting the secondary antibodies far enough.

2. What are some causes of weak signal?

There is insufficient antigen present and an amplification protocol may be needed.
Change from a more sensitive detection method to a less sensitive detection method.
The primary antibody is diluted too far.
Enzyme activity is inhibited.
The secondary antibody does not recognize certain primary antibodies well.
The secondary antibody cross-reacts with immunoglobulins in the diluent.

3. What diluent is recommended for the secondary antibody?

The antibodies can be diluted in buffers such as PBS or TBS. A detergent such as Tween 20 (0.05% v/v) can be included to reduce non-specific binding. We recommend making the working dilution fresh on the day of use. Carrier proteins such as BSA or normal serum are not necessary, and in some cases can increase background staining due to protein-protein interactions.
Read more about diluents in our guide to blocking, controls and diluents.

4. What blocking buffer is recommended?

For most applications, we recommend blocking with normal serum (5% v/v) from the same species as the host of the secondary antibody. Other commonly used reagents such as BSA and non-fat dry milk are suitable for some applications, but may increase background if the secondary antibody is directed against goat, sheep, or horse.
Read more about blocking in our guide to blocking, controls and diluents.

5. What incubation time is recommended for the secondary antibody?

Incubation times of 30-60 minutes are recommended for surface interactions such as ELISA or Western blotting. For staining tissue, incubation times should be increased as necessary to allow for tissue penetration. Total wash time should be approximately equal to incubation time to allow unreacted antibody to diffuse out.

Antibody Structure Questions

1. What is an antibody subclass?

Immunoglobulin classes (IgA, IgD, IgE, IgG, IgM in mammals) are defined by the structure of their heavy chains. Minor differences on heavy chains are found between subclasses of each class. Jackson ImmunoResearch offers polyclonal antibodies that are specific for mouse subclasses IgG1, IgG2a, IgG2b, IgG2c, and IgG3. We also have antibodies that are specific for camelid subclasses IgG 2+3. We currently do not have subclass-specific antibodies against other species.
Download Immunoglobulin Structure and Function Poster.

2. What are the differences between Fab, Fc, and F(ab')2 fragments?

These products are the result of proteolytic digestion of IgG. Digestion with papain results in Fab and Fc fragments, and digestion with pepsin yields F(ab’)2 fragments.

3. What is an antibody isotype?

Isotypes are distinct forms of antibody heavy and light chains, and may refer to class or subclass. Light chain isotypes are kappa and lambda, and heavy chain isotypes are alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG), mu (IgM). The heavy chains may be further differentiated as subclasses, e.g. gamma 1 (IgG1).

4. What is an isotype control?

Isotypes are distinct forms of antibody heavy and light chains, and may refer to class or subclass. Light chain isotypes are kappa and lambda, and heavy chain isotypes are alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG), mu (IgM). The heavy chains may be further differentiated as subclasses e.g. gamma 1 (IgG1).
An isotype control is a negative control that verifies specific binding of either a primary or secondary antibody. The isotype control is the same class or subclass as the experimental antibody, but is not specific for the antigenic target. Isotype controls may be purified IgG from normal serum of the species host of the experimental antibody; or in the case of monoclonal antibodies may be monoclonals of the same subclass, directed against an irrelevant antigen. Isotype controls for unlabeled antibodies are unlabeled, and controls for labeled antibodies are labeled with the same reporter molecule (fluorophore, enzyme, or biotin) as the experimental antibody.


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10 tips for better blots

Western blotting

Western blotting is a technique used to confirm the presence of target proteins and peptides from complex mixtures. Proteins are Read More...

How to select a Secondary Antibody

Choosing the right affinity-purified secondary antibody for your application The following information details how to use the Product Filter on Read More...

Mouse Subclass Specific Secondary Antibodies

Anti-Mouse IgG Subclass Specific Secondary Antibodies Mouse IgG subclasses Mice express 4 of the 5 available IgG subclasses making up Read More…

Light Chain Specific Secondary Antibodies

Western blotting after Immunoprecipitation For researchers who perform Western blotting following immunoprecipitation, antibodies specific for light chains or Fc fragments Read More…

Primary Antibodies for Signal Enhancement

Additional Antibody Specificities Anti-Biotin, Anti-Fluorescein and Anti-Digoxin antibodies Monoclonal Mouse Anti-Biotin, Anti-Fluorescein and Anti-Digoxin are available in a wide range Read More…

Cross-Adsorbed Secondary Antibodies

Cross-adsorbed (min X) secondary antibodies and cross-reactivity Immunoglobulins from different species share similar structures. Secondary antibodies raised against one species Read More...

JIR Product Descriptions

Complete Product Description For Ordering Purposes For a complete description of the product, please use the following format to avoid Read More...

FLUOROPHORE SELECTION TIPS AND TOOLS

Fluorophore selection and panel building – Spectra Viewer

Fluorescent probes or fluorophores (fluorescent dyes or proteins) are coupled to a secondary antibody or streptavidin to allow visualization of Read More…

NEW Secondary Antibodies For VHH Discovery

Secondary Antibodies For VHH Discovery

JIR presents Anti-Alpaca IgG, Subclass and VHH domain secondary antibodies to optimize VHH (single domain) antibody development and application. Pathway Read More…

Anti-Camelid Secondary Antibodies

Anti-Camelid Secondary Antibodies

At Jackson ImmunoResearch, our mission is to develop the widest range of high quality secondary antibodies for the life science Read More…

Chromogenic Detection for Western Blot, IHC, and ELISA

Chromogenic substrates are used in colorimetric detection. They are simple and easy to use. Suitable for most immunotechniques – from Read More…

Colorimetric Western blotting

Colorimetric detection is an economical and simple method for the detection of analyte when Western blotting. Enzyme reporters conjugated to Read More…

Chemiluminescent Western blotting

Chemiluminescent Western blotting is a highly sensitive protein detection method. The broad dynamic range allows analyte detection over a wide Read More…

Fluorescent Western blotting

Fluorescent Western blotting can offer many advantages to an already robust protein detection technique. Secondary Antibodies are conjugated to a Read More…

Conjugates for Western blotting

3 methods of detection are available for Western blotting: colorimetric, chemiluminescent and fluorescent. Each detection method can offer advantages, in Read More…

Direct and Indirect Western blotting

Western blotting is a robust technique employing antibodies to detect proteins immobilized on a blotting membrane after separation by electrophoresis. Read More…

First Class Secondary Antibodies

SelectScience® Gold Seal for Quality awarded to JIR

The Gold Seal of Quality has been awarded to Jackson ImmunoResearch Laboratories Inc. for consistently receiving multiple positive reviews on Read More…

Distinguish cell types from primary cultures

“When labeling for more than one target with antibodies from closely related species, you really need to be certain your Read More…