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Antibody Technical FAQ


General questions

  1. In general, what is the recommended working concentration of your primary antibody?

    Western blot test: 0.1-1 μg/ml
    IHC, ICC, FACs and IP test: 1-5 μg/ml
    Elisa test: 0.05-0.2 ug/ml

  2. Do you have any antibody products mainly designed for pharmaceutical manufactures?

    Yes.


  3. What are the reasons for high background in WB test?

    1. Primary antibody concentration may be too high.
    2. The blocking of non-specific binding may be insufficient.
    3. Non-specific binding of antibody.
    4. Too much sample may be loaded.
    5. Secondary antibody concentration may be too high.

  4. The protein band in the western blot is different from the predicted molecular weight of the protein. What might be the possible reason?

    1. Post-tanslational modification, such as phosphorylation, glycosylation, etc. All of these may increase the protein molecular weight.
    2. Post-translational cleavage: for protein processing, i.e. cleavage from a precursor to a mature active form, such as occurs with a caspase precursor.
    3. Splice variant: isoforms from alternative splicing.
    4. Relative charged-amino acid composition (the ratio of both charged and uncharged amino acids)

  5. When I analyze cytoplasmic protein detection by FACS, the result is negative. However the result is positive when analyzing by Western Blot showing a band with the right M.W. What is the possible reason?

    1. Antibody working concentration is low
    2. Cell membrane was not penetrated sufficiently
    3. Antibody incubation time is too short
    4. Detection of FACS excitation wavelength is not consistent with fluorescence-labeled antibody excitation wavelength.

  6. In general, how should I preserve the antibody?

    Please read and carefully follow the manual instructions on how to preserve each antibody product.

    Typically, store antibodies at 2-8C for short term storage and store at -20C for long term storage. Aliquot and store before use to avoid repeated freeze-thaw cycles.

    The following antibody products need to be stored in the dark.

    1. THE™ HA Tag Antibody [iFluor 555], mAb, Mouse
    2. THE™ DYKDDDDK Tag Antibody [iFluor 647], mAb, Mouse
    3. THE™ V5 Tag Antibody [iFluor 488], mAb, Mouse
    4. THE™ His Tag Antibody [FITC], mAb, Mouse

  7. How do you guarantee the quality of the antibodies?

    To ensure antibody quality between different batches, our QA tests and compares sensitivity and specificity between batches

  8. Should I use Monoclonal Antibodies or Polyclonal Antibodies?

    Both Polyclonal and Monoclonal antibodies have their own advantages which make them useful for different applications.

    1. Polyclonal Antibodies

      Large quantities of polyclonal antibody are relatively quick and inexpensive to produce compared to monoclonal antibodies. They are non-specific in that they are capable of recognizing multiple epitopes on any one antigen.

      1. Advantages

        • Can help increase the WB signal as the antibody will bind to more than one epitope.
        • Due to recognition of multiple epitopes, polyclonal antibodies can give better results in IP/ChIP assays.
        • More tolerant of minor changes in the antigen, e.g., polymorphism, heterogeneity of glycosylation, or slight denaturation.
        • Useful when the nature of the antigen is unknown
        • Inexpensive to produce and timeline is short

      2. Disadvantages

        • More prone to batch to batch variability.
        • Multiple epitopes make it important to check immunogen sequence for any potential cross-reactivity.

    2. Monoclonal Antibodies

      1. Advantages

        • Compared to polyclonal antibodies, homogeneity of monoclonal antibodies is very high.
        • If experimental conditions are kept constant, results from monoclonal antibodies will be highly reproducible between experiments.
        • All batches will be identical and specific to just one epitope which is a particular advantage when manufacturing procedures must be standardized e.g. clinical tests and therapeutic treatments.
        • The high specificity of monoclonal antibodies decreases background noise and cross-reactivity, helps provide reproducible results and ensure efficiency in affinity purification.
        • Specific antibody characteristics can be identified and selected e.g. sensitivity requirements and cross reactivity levels can be specified and screened to identify cell lines exhibiting the required characteristics.
        • Hybridomas provide an immortal cell line with the ability to produce unlimited quantities of highly specific antibodies.

      2. Disadvantages

        • Antibodies may be too specific (e.g. less likely to detect across a range of species)
        • More vulnerable to the loss of epitope through chemical treatment of the antigen than polyclonal antibodies. This can be offset by pooling two or more monoclonal antibodies to the same antigen.
        • Expensive to produce and timeline is long for hybridomas

Epitope Tag Antibody & THE™ Elite Antibody FAQ

  1. Will using a brighter fluorophore allow better detection of a molecule on a cell that is in low abundance?

    The brightness of fluorophore and the antibody's sensitivity are two factors that influence detection sensitivity. THE™ Elite antibody series have the best sensitivity on the market. The brighter the fluorophore is, the better the overall detection. GenScript's iFluor antibody series are newly developed fluorescent compounds conjugated to THE™ Elite antibody series. Compared with traditional fluorescent dyes, the iFluor series are brighter and have better stability.

  2. Which antibody should I use to detect a very small amount of His protein in a Flow Cytometry assay?

    GenScript has four types of fluorophore conjugated His-tag antibodies for FACS analysis, including A01620 (FITC), A01800 (iFluor488), A01801 (iFluor555) and A01802 (iFluor 647). The iFluor series are recommended for FACS analysis because they have a higher sensitivity than FITC conjugated antibody. The following is the wavelength range of GenScript's iFluor antibodies:

    Cat. No.
    Conjugate
    Ex (nm)
    Em (nm)
    A01800
    iFluor 488
    491
    514
    A01801
    iFluor 555
    559
    569
    A01802
    iFluor 647
    654
    674

    According to our FACS analysis results, the sensitivity of A01802 (THE™ His Tag Antibody [iFluor 647], mAb, Mouse) is slightly higher than the A01800 (THE™ His Tag Antibody [iFluor 488], mAb, Mouse). If the His Tag number is low, A01802 is recommended.

  3. For A00186 (THE™ His Tag Antibody, mAb, Mouse), what is the working concentration range for Immunofluorescence?

    For A00186, a starting point of 1 µg/ ml is recommended.

  4. We are using your ELISA kit L00436 (His Tag ELISA Detection Kit) and we like to use the same anti-His monoclonal antibody for western blot. What is the Catalog No. for the mAb used in the L00436 ELISA kit? What is its recommended concentration for WB?

    The catalog No. is A00186 for the anti-His mAb in the L00436 ELISA Kit. The recommended concentration for WB is 0.1-0.2 µg/ml.

  5. I need a rabbit anti-His tag antibody which can recognize a His-tag fused on an internal site of a protein. I want to perform Flow Cytometry with this antibody. Which antibody should I use?

    Catalog No. A00174 is a rabbit anti-His tag antibody. It has been tested for internal His-tag recognition, but not for Flow Cytometry.

    For Flow Cytometry, the following antibodies are recommended. All of them can recognize His-tag fused on the internal site of a protein.


  6. Questions about A01795 (THE™ PEG Antibody, mAb, Mouse)

    1. Can A01795 (THE™ PEG Antibody, mAb, Mouse) recognize PEG5?

      We have not yet tested PEG5. We tested PEG12 with positive results. One of our clients tested PEG2 and observed good results.

    2. Can I use A01795 (THE™ PEG Antibody, mAb, Mouse) in building an affinity column.

      In most cases it should be OK to conjugate an antibody (IgG/IgM/IgY) to agarose resin. Our PEG antibody could be used for this conjugation.

    3. Do you have any information on antibody stability after reconstitution? I'm concerned that an IgM antibody may have fragmented during the lyophilization process or became aggregated upon reconstitution.
    4. As mentioned in the datasheet, THE™ PEG Antibody, mAb, mouse should be stored in its lyophilized form until use. It remains stable in lyophilized form if stored at -20°C or below. The reconstituted antibody can be stored for 2-3 weeks at 2-8°C or for up to 12 months at -20°C or below. Avoid repeated freeze-thaw cycles.

      Reconstitute the lyophilized product with deionized water (or equivalent) to make an antibody concentration of 0.5 mg/ml. For long-term storage, it is recommended to add glycerol to a final concentration of 50% (v/v) in the reconstituted antibody solution.

  7. I've purchased A01428-100 (THE™ DYKDDDDK Tag Antibody [HRP], mAb, Mouse). I was wondering if the presence of HRP will interfere with secondary antibody binding (either Goat anti-Mouse secondary or Protein G secondary).

    When using [HRP] tagged primary antibodies there are some variables that may affect its binding with a secondary antibody.

    1. Steric hindrance caused by HRP
    2. The binding sites of antibodies may be occupied by HRP
      Therefore, we recommend using the unconjugated antibody A00187 THE™ DYKDDDDK Tag Antibody, mAb, Mouse

Secondary Antibody FAQ

  1. Is Catalog No. A00166 (Goat Anti-Human IgG Antibody (H&L) [HRP], pAb) specific for human IgG. Does it cross-react with non-human primates or rodents?

    Catalog No. A00166 (Goat Anti-Human IgG Antibody (H&L) [HRP], pAb) recognizes human IgG, but it has not yet been tested for cross-reactivity with other species.

  2. Does Catalog No. A00160 (Goat Anti-Mouse IgG Antibody (H&L) [HRP], pAb) cross react with rabbit?

    Catalog. No A00160 (Goat Anti-Mouse IgG Antibody (H&L) [HRP], pAb) has not yet been tested for cross-reactivity with rabbit.

Primary Antibody FAQ

 
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