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Antibody Technical FAQ

• General questions
• Epitope Tag Antibody & THE™ Elite Antibody FAQ
• Secondary Antibody FAQ
• Primary Antibody 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 μg/ml

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

   Yes.

   • THE™ cAMP Antibody, mAb, Mouse
   • THE™ ADP Antibody, mAb, Mouse
   • THE™ PEG Antibody, mAb, Mouse
   • Protein A ELISA Kit
   
   
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 亚博的官网地址iant: 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℃ for short term storage and store at -20℃ 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 亚博的官网地址iability.
• 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:

   WhichantibodyshouldIusetodetectaverysmallamountofHisproteininaFlowCytometryassay?

   Em(nm)

   491

   iFluor488

   674

   GenScripthasfourtypesoffluorophoreconjugatedHis-tagantibodiesforFACSanalysis,includingA01620(FITC),A01800(iFluor488),A01801(iFluor555)andA01802(iFluor647).

   Conjugate

   559

   A01800

   TheiFluorseriesarerecommendedforFACSanalysisbecausetheyhaveahighersensitivitythanFITCconjugatedantibody.ThefollowingisthewavelengthrangeofGenScript'siFluorantibodies:

   569

   iFluor555

   AccordingtoourFACSanalysisresults,thesensitivityofA01802(THE™HisTagAntibody[iFluor647],mAb,Mouse)isslightlyhigherthantheA01800(THE™HisTagAntibody[iFluor488],mAb,Mouse).IftheHisTagnumberislow,A01802isrecommended.

   654

   514

   A01802

   Cat.No.

   A01801

   Ex(nm)

   iFluor647

   1. The MW of the target protein is about 18 kDa, so why is the band in the WB test picture almost 60 kDa.

      The target protein tested is 57 KDa because it is the recombinant protein, not the naturally occurring protein with the molecular weight of 18 KDa.

   2. What is the fusion partner?

      From the COA file, you can see that the fusion partners for this protein are calbindin, Trx tag and Strip-Ⅱtag.

   3. Is there any cross-reactivity between the fusion partner and PIN1?

      According to multiple sequence alignment results, there is only one sequence in the recombinant protein with the peptide antigen for immunization. Based on these results there should be no cross-reactivity between the fusion partner and PIN1.

   4. How is PIN1 fused to its partner?

      PIN1 is fused to its partner at the N terminal.