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Antibody FAQs

How will I receive my antibody?

When you order an antibody from us, it will be shipped at room temperature in a stable lyophilized form.

What is a blocking peptide and how is it used?

The blocking peptide is the antigen we use for immunization during antibody generation so that we can create an antigen-specific antibody. You can use this blocking peptide as a negative control alongside other controls in an immunoassay to give some insight into antibody specificity, but not selectivity.

It’s important to note that the blocking peptide is not an effective control for use with live cells. If you need a control for flow cytometry, we recommend an isotype antibody control instead.

For more details, see our guide to blocking peptides.

How do I avoid cross-reactivity between close members of the same protein family?

We carefully choose the epitope for immunization, taking into account two main parameters: immunogenicity and specificity of the epitope.

Before immunization, we run a search to verify that the chosen epitope has maximum homology with other species and minimum homology among members of the same family or other proteins. After immunization and collection of sera, the antibodies are affinity-purified on immobilized antigens (the injected peptides).

This means you shouldn’t have to worry about your antibody cross-reacting with close members of the same protein family.

What is the antibody isotype of rabbit polyclonal antibodies?

Affinity-purified rabbit polyclonal antibodies are mainly IgGs.

How do I reconstitute the antibody?

Your antibody comes as a lyophilized powder in phosphate-buffered saline (PBS), (pH 7.4), with 1% BSA, and 0.05% NaN3. Before you reconstitute the antibody, we recommend you briefly centrifuge the vial to ensure all of the antibody is at the bottom of the tube. You can then reconstitute the antibody in double-distilled water (DDW) or any other water that you prefer (such as UltraPure DNase/RNase-Free Water). Note: the volume of water that you should add to reconstitute the antibody is shown on the vial. After reconstitution, you can further dilute the antibody to the recommended working concentration with any desired buffer.

How do I store my antibody and how long will it last?

In general, upon arrival, the lyophilized antibody (before reconstitution) should be kept at -20°C until use. If kept as a lyophilized powder at -20°C, you can store it safely for several months if you avoid repeated freeze-thaw cycles.

After reconstitution, you should sire it at 4°C, and it should retain its reactivity for approximately one week. For long-term storage, aliquot your antibody and store at -20°C until use. The aliquoted samples stored at -20°C should retain their reactivity for approximately 1 year if handled well.

We do not recommend storing the antibodies at -80°C.

What is the role of bovine serum albumin (BSA) and sodium azide (NaN3) in the antibody formulation?

BSA serves as an effective stabilizer (that supplies a high protein concentration environment for the antibody) and minimizes the loss of antibody due to non-specific binding to the tube. Meanwhile, the NaN3 functions as a bacteriostatic preservative, which prevents microbial contamination.

If you need an antibody without BSA or NaN3, just let us know and we can prepare a carrier- and/or preservative-free version for you.

What is an ‘extracellular’ antibody?

The “extracellular” part in the antibody title simply tells you the chosen epitope is facing the extracellular space. Antibodies that don’t have “extracellular” in the product name are antibodies that target an epitope that faces the intracellular space of the cell.

Antibodies that recognize extracellular domains of proteins are great tools for live-cell imaging and live-cell flow cytometry since there’s no need for permeabilization and fixation.

Are different lots of the same antibody just different bleeds from the same animal or are they from different animals?

Each lot can be from different bleeds from the same animal and, of course, from different animals. In any case, all our antibodies are affinity-purified in columns with immobilized peptide antigen. Additionally, all new lots are tested in a western blot in parallel with a previous lot, to ensure the best product possible by minimizing lot to lot inconsistencies.

The peptide (used for immunization to generate the antibody) sequence on your datasheet does not include a Cysteine residue but it has a (C) in parentheses. What does it mean?

It is customary to denote any changes or additions to the original peptide sequence with parentheses. We add a Cysteine (Cys) to all peptides so they can be covalently linked to a carrier protein for immunization and the solid phase used for affinity purification of the antibody.

What are ATTO Fluor-conjugated antibodies?

ATTO Fluor-conjugated antibodies are primary antibodies that contain an ATTO Fluor dye (fluorophore) directly attached to them, which eliminates the need for a secondary antibody.

ATTO Fluor dyes have strong absorption (high extinction coefficient), high fluorescence quantum yield, and high photostability. They are analogous to the Alexa Fluor dyes and are comparable to other currently available fluorescent technologies. The ATTO Fluor dyes are especially suited for applications that require simultaneous labeling of different markers.

You can read about ATTO Fluor dyes in our short application guide.

Are antibodies conjugated to dyes light-sensitive?

Yes. Our primary antibodies that have been conjugated to a dye, such as ATTO Fluor, are sensitive to light. You should keep them covered or in the dark as much as possible. The vials can be kept in light-proof containers or wrapped in foil during experiments to help shield them from the light.

What is a fusion protein?

Fusion proteins are made from at least two joined domains – encoded by different genes – so that they are transcribed and translated as a single unit, to yield a single polypeptide. You can use fusion protein in the form of affinity tags to help with protein purification, where you take a protein you can easily purify by affinity chromatography and fuse it to the protein you want to research. A common affinity tag is glutathione S-transferase (GST). In this case, you would insert GST’s DNA sequence alongside the DNA of the protein of interest and express it in bacteria, resulting in a GST-tagged fusion protein. This fused protein would then need to undergo strict purification methods from other bacterial proteins and debris. You then confirm the identity and purity of the fused protein by DNA sequence and SDS-PAGE.

Do you have antibodies generated against fusion proteins?

Yes, we do. We use GST-tagged proteins to immunize hosts like rabbits or guinea pigs to generate antibodies against them. The host’s immune system then generates antibodies against both the GST tag and the specific protein attached to it. To isolate these specific antibodies, we first deplete the collected sera of anti-GST antibodies by affinity chromatography and then purify the antibodies directed against the specific protein on an immobilized antigen.

For further assistance, please contact us at [email protected].