Antibody FAQs

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

 

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Why do different lots of the same antibody give different western blot results?

Alomone Labs antibodies are mostly polyclonal. Differences between lots may occur. Our protocols are designed to minimize these differences but we cannot completely avoid them. Each lot is purified by affinity column. The antibodies undergo quality control using western blot and are released only after satisfactory results are obtained. Experimental conditions, such as concentration, incubation time, exposure time, etc, may change between different batches and require further calibration.

Why are there differences between the detected MW band in a western blot and the calculated one?

The calculated MW of a protein is based only on its amino acid sequence and does not account for posttranslational modifications such as glycosylation, phosphorylation, etc. Therefore, the protein might be detected at a different MW.

What is the negative control antigen and how is it used?

The negative control antigen is the antigen used for the immunization of the rabbits in order to obtain the antigen-specific antibody. The negative control antigen can be used as a negative control in a western blot in order to verify the specificity of the bands detected with the antibody.

See “Antigen Manipulation” in Protocols.

How do you 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.

The antibodies are affinity purified on immobilized antigens (the injected peptides).

What is the antibody isotype of rabbit polyclonal antibodies?

Affinity purified rabbit polyclonal antibodies are mainly IgGs.

How do I reconstitute the antibody?

The antibody is supplied as lyophilized powder in phosphate buffered saline (PBS), pH 7.4, 1% BSA, 0.05% NaN3. It should be reconstituted in double distilled water (DDW), depending on the sample size. After reconstitution the antibody can be further diluted to the recommended working dilution with the desired buffer.

What are antibodies conjugated to ATTO dyes?

ATTO dyes are characterized by strong absorption (high extinction coefficient), high fluorescence quantum yield, and high photo-stability. They are analogous to the Alexa dyes and are comparable to other fluorescent technology in the market. ATTO dyes are especially suited for applications that require simultaneous labeling of different markers.

What are the advantages of extracellular antibodies?

Antibodies recognizing extracellular domains of proteins are great tools for Live Cell Imaging and Live Cell Flow Cytometry. No need for permeabilization and fixation.