Overview

  • Product name
  • Description
    Rabbit polyclonal to NFYB
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, IP, ELISA, EMSAmore details
  • Species reactivity
    Reacts with: Mouse, Human
  • Immunogen

    NF-Y (B subunit specific) peptide corresponding to a region near the N-terminus of the human protein conjugated to Keyhole Limpet Hemocyanin (KLH). The sequence is: QQI SGV QQI QFS

  • Positive control
    • CHO-7 cells that were cultured in the presence of cholesterol.

Properties

Applications

Our Abpromise guarantee covers the use of ab6559 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB 1/500. Predicted molecular weight: 23 kDa.
IP Use at an assay dependent dilution.
ELISA 1/4000.
EMSA Use 2µl for 104 cells.

Target

  • Function
    Stimulates the transcription of various genes by recognizing and binding to a CCAAT motif in promoters, for example in type 1 collagen, albumin and beta-actin genes.
  • Sequence similarities
    Belongs to the NFYB/HAP3 subunit family.
  • Domain
    Can be divided into 3 domains: the weakly conserved A domain, the highly conserved B domain thought to be involved in subunit interaction and DNA binding, and the Glu-rich C domain.
  • Cellular localization
    Nucleus.
  • Information by UniProt
  • Database links
  • Alternative names
    • CAAT box DNA binding protein subunit B antibody
    • CAAT box DNA-binding protein subunit B antibody
    • CBF A antibody
    • CBF B antibody
    • CCAAT binding transcription factor subunit A antibody
    • HAP 3 antibody
    • HAP3 antibody
    • NF Y protein chain B antibody
    • NF YB antibody
    • NF-YB antibody
    • NFYB antibody
    • NFYB_HUMAN antibody
    • Nuclear transcription factor Y beta antibody
    • Nuclear transcription factor Y subunit B antibody
    • Nuclear transcription factor Y subunit beta antibody
    • Transcription factor NF Y B subunit antibody
    see all

Images

  • EMSA was performed with a double-stranded, biotin-labeled oligonucleotide in the presence of murine ES cell nuclear extracts and increasing amounts of anti-NFYB antibody (ab6559; 2 µg or 6 µg) or unrelated antibody.

References

This product has been referenced in:
  • Braden CR & Neufeld TP Atg1-independent induction of autophagy by the Drosophila Ulk3 homolog, ADUK. FEBS J 283:3889-3897 (2016). Read more (PubMed: 27717182) »
  • Huang Y  et al. Phospho-?Np63a is a key regulator of the cisplatin-induced microRNAome in cancer cells. Cell Death Differ 18:1220-30 (2011). WB ; Human . Read more (PubMed: 21274007) »
See all 3 Publications for this product

Customer reviews and Q&As

1-2 of 2 Abreviews or Q&A

Question

1. Antibody storage conditions (temperature/reconstitution etc) -20oC 2. Please describe the problem (high background, wrong band size, more bands, no band etc). major band is wrong size 50kd 3. On what material are you testing the antibody in WB? Species: 293T human Cell extract or Nuclear extract:Cell extract Purified protein or Recombinant protein: 3. The lysate How much protein was loaded:100ug What lysis buffer was used:RIPA What protease inhibitors were used:no What loading buffer was used: 3X loading dye from moleunlar cloning Did you heat the samples: temperature and time:95 oC , 10min 4. Electrophoresis/Gel conditions/ Transfer conditions Reducing or non reducing gel: reducing gel Gel percentage :15% Transfer conditions: 150V 2hr Buffer:5% milk Blocking agent: milk, BSA, serum, what percentage:5% Incubation time:over night Incubation temperature:4 oC 6. Primary Antibody Specification (in which species was it raised against):rabbit At what dilution(s) have you tested this antibody:1:1000 What dilution buffer was used:5% milk Incubation time:3hr Incubation temperature:RT What washing steps were done:1X TBST 3 times 7. Secondary Antibody Specification (in which species was it raised against)?rabbit At what dilution(s) have you tested this antibody:1:2000 Incubation time1hr Wash steps:1X TBST 3times Do you know whether the problems you are experiencing come from the secondary?the secondary ab is ok 8. Detection method ECl 9. Background bands Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a "No primary" control): Is the blocking step sufficient? Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) At what size are the bands migrating? Could they be degradation products of your target? Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient) 11. Did you apply positive and negative controls along with the samples? Please specify. 10. Optimization attempts How many times have you tried the Western?Twice Do you obtain the same results every time e.g. are background bands always in the same place? yes What steps have you altered? 1o ab incubation time

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Answer

I'm sorry to hear you are having problems with ab6559. The protein NF-YB is know to function as a heterotrimeric transcription factor when bound to NF-YA and NF-YC. It is therefore possible that the large protein observed is made of NF-YB and other parts of the heterotrimer (NF-YB is 22.8kDa, NF-YA is 36kDa, NF-YC is 50.3kDa). I would recommend the following modifications to your protocol: -use protease inhibitors in the lysis buffer -make sure the loading buffer is reducing (i.e. containing SDS, beta mercaptoethanol, DTT) -load less protein per well (25-40ug) -block the membrane in milk (you may wish to try BSA 5% as well) for 1hr at RT -incubate the primary antibody 1:1000 at 4C overnight in TBST (no milk) -incubate the secondary antibody in TBST Please note that the protein is found in the nucleus and should the band be due to non-specific binding of the antibody you may need to concentrate your samples by nuclear extraction prior to SDS separation. I hope these suggestions will help you, please do not hesitate to contact us again if you still experience problems,

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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