Documents & Support

How can I determine the degree of protein labeling? Product FAQ

Answer

To quantitate biotin, we offer two kits as follows:


  • Biotin Quantitation Kit (Cat. No. 28005): With this kit, a solution containing the biotinylated protein is added to a mixture of HABA reagent (4’-hydroxyazobenzene-2-carboxylic acid and avidin. Because of its higher affinity for avidin, biotin displaces the HABA and the absorbance at 500nm decreases proportionally. 
  • Fluorescence Biotin Quantitation kit (Cat. No. 46610): This microplate-based biotin assay is easy to perform by adding the supplied fluorescent reporter to the biotinylated samples and diluted biocytin standards. The avidin fluoresces when the weakly interacting HABA (4’-hydroxyazobenzene-2-carboxylic acid) is displaced by the biotin. The amount of biotin is determined by comparing the sample's fluorescence to the biocytin standard curve. This assay requires must less sample volume than the microplate colorimetric HABA assay and is much more sensitive

  • To determine the dye-to-protein ratio after fluorophore conjugation, absorbance readings of the protein:dye conjugate are taken and the molar ratio can then be calculated. Please go to this Tech Tip (https://tools.thermofisher.com/content/sfs/brochures/TR0031-Calc-FP-ratios.pdf")for more information

    Answer Id:: E15705

    Was this answer helpful?

    Yes No

    Thank you for your response

    How can I biotinylate my probe for the North2South Chemiluminescent Hybridization and Detection Kit? Product FAQ

    Answer

    Biotinylated DNA probes can be generated with the North2South Biotin Random Prime DNA Labeling Kit (Cat. No. 17075). Probes may also be generated by PCR, random primed labeling or in vitro transcription. Biotinylated probes made with reagents and kits from other companies are compatible.

    Answer Id:: E8443

    Was this answer helpful?

    Yes No

    Thank you for your response

    How do I prepare my labeled RNA probe for my RNA Protein Pull-Down Assay? Product FAQ

    Answer

    Labeled RNA probes can be purchased commercially or generated through either run-off in vitro transcription reactions with biotinylated nucleotides or through enzymatic ligation of biotin tags to the 3' terminus of an RNA strand using the RNA 3' End Biotinylation Kit (Cat. No. 20160) or RNA 3’ End Desthiobiotinylation Kit (Cat. No. 20163). Desthiobiotinylated cytidine is utilized for the pulldown assay to enable a gentle elution to capture protein-RNA complexes. Reagents and conditions are included in the kit to relax the RNA for better labeling efficiency. Additionally, suggestions are included in the instruction manual for optimization of the ligation reaction. An RNA control is included in the kit to assess kit labeling efficiency.

    Answer Id:: E15499

    Was this answer helpful?

    Yes No

    Thank you for your response

    The far-western blot signal I obtained was very low, how can I improve this? Product FAQ

    Answer

    The following are the reasons and remedies for low signal in far-western blot application:

    1. The streptavidin-HRP is too dilute or has low activity: Try a 2- to 5-fold higher concentration or use fresh streptavidin-HRP.
    2. The biotinylated-protein probe is too dilute: Try a 2- to 5-fold higher biotinylated-protein probe concentration. 
      Note: Alter the biotinylated “probe” (bait) protein concentration only if adjusting the streptavidin-HRP concentration has no effect or results in high background. 
    3. The probe protein not sufficiently biotinylated: Biotinylate the probe (bait) protein again with a higher molar excess of biotinylation reagent to protein; the mole-to-mole ratio or degree-of-labeling of biotin may be determined using HABA (Cat. No. 28010) or the Fluorescence Biotin Quantitation Kit (Cat. No. 46610) each time the probe is biotinylated to make sure that consistent biotinylation level is obtained.
      Note: There can be a fine line between sufficient labeling for detection and over labeling with subsequent steric hindrance and loss of signal. Optimizing the labeling reaction prior to the far-Western can reduce downstream optimization, which can use up precious samples.
    4. The biotinylated protein probe interacts weakly with “prey” protein: Use native conditions to separate samples, as denaturing conditions may alter the proteins such that they do not interact with the “probe” during electrophoresis and transfer.
    5. It is possible that a change in three-dimensional structure took place due to the protein absorbing into the membrane, changing the interaction site. In this case, far-western analysis may not be an appropriate approach for detection of a protein-protein interaction and cross-linking or label transfer may be other viable options to confirm the interacting partnership. 
    Answer Id:: E15641

    Was this answer helpful?

    Yes No

    Thank you for your response

    What are the total RNA input requirements for GeneChip miRNA 3.1 Array Strip? Product FAQ

    Answer

    If you are using a FlashTag Biotin HSR RNA Labeling Kit for the GeneChip miRNA 3.1 Array Strip, we recommend staying within the range of 130-1000 ng input total RNA.

    Answer Id:: E17996

    Was this answer helpful?

    Yes No

    Thank you for your response

    Do we have to use any Affymetrix Reagents with our assay? Product FAQ

    Answer

    GeneChip Hybridization Control Kit 30 reactions
    GeneTitan Hybridization, Wash, and Stain Kit for miRNA Array Plates 96 reactions
    GeneTitan Hybridization Module for miRNA Plates 96 reactions
    Affymetrix FlashTag Biotin HSR RNA Labeling Kit 10 reactions
    Affymetrix FlashTag Biotin HSR RNA Labeling Kit 30 reactions

    Answer Id:: E14203

    Was this answer helpful?

    Yes No

    Thank you for your response

    Click Chemistry Reagents Manual / Product Insert

    • Version: 08-05-2009
    Catalog #

    Product Information Sheet: ProtoArray Human Protein Microarray for for Protein-Protein Interaction (PPI) Manual / Product Insert

    • Version: Rev B (17 March 2017)
    Catalog #
    • PAH0525101(Discontinued)
    • PAH05251020(Discontinued)

    Protocol: MessageAmp™ II-96 Kit (English ) Manual / Product Insert

    • Version: 16 Feb 2011
    Catalog #

    Data Sheets: Standardized Assays and Reagents for GeneChip Expression Analysis Product Literature

    User Guide: EZ-Link NHS-SS-PEG4-Biotin Manual / Product Insert

    • Version: MAN0016366 Rev. A.0 (16Nov2016)
    Catalog # 21442

    Dynabeads® FlowComp™ Mouse CD4+CD25+ Treg Cells Manual / Product Insert

    • Version: 002
    Catalog # 11463D

    User Guide: AminoLink Reductant Manual / Product Insert

    • Version: Jan. 2015
    Catalog # 44892
    Results per page
      spinner