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  1. Link

    products/primary-antibodies/hec1hec-antibody-9g3-ab3613.pdf

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Anti-HEC1/HEC antibody [9G3] (ab3613)

  • Datasheet
Reviews (11)Q&A (7)References (102)

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Immunocytochemistry/ Immunofluorescence - Anti-HEC1/HEC antibody [9G3] (ab3613)
  • Western blot - Anti-HEC1/HEC antibody [9G3] (ab3613)
  • Immunocytochemistry/ Immunofluorescence - Anti-HEC1/HEC antibody [9G3] (ab3613)
  • Immunoprecipitation - Anti-HEC1/HEC antibody [9G3] (ab3613)
  • Immunocytochemistry/ Immunofluorescence - Anti-HEC1/HEC antibody [9G3] (ab3613)
  • Western blot - Anti-HEC1/HEC antibody [9G3] (ab3613)
  • Flow Cytometry - Anti-HEC1/HEC antibody [9G3] (ab3613)
  • Immunocytochemistry/ Immunofluorescence - Anti-HEC1/HEC antibody [9G3] (ab3613)

Key features and details

  • Mouse monoclonal [9G3] to HEC1/HEC
  • Suitable for: Flow Cyt, IP, WB, ICC/IF
  • Reacts with: Human, Pig
  • Isotype: IgG2a

You may also be interested in

Protein
Product image
Recombinant Human HEC1/HEC protein (ab131907)
Biochemical
Product image
SAG (Smo agonist), Smoothened agonist (ab142160)
Secondary
Product image
Goat Anti-Mouse IgG H&L (HRP) (ab205719)

View more associated products

Overview

  • Product name

    Anti-HEC1/HEC antibody [9G3]
    See all HEC1/HEC primary antibodies
  • Description

    Mouse monoclonal [9G3] to HEC1/HEC
  • Host species

    Mouse
  • Tested applications

    Suitable for: Flow Cyt, IP, WB, ICC/IFmore details
  • Species reactivity

    Reacts with: Human, Pig
  • Immunogen

    Recombinant fragment corresponding to Human HEC1/HEC aa 56-642.
    Database link: O14777

  • Positive control

    • WB: HeLa whole cell lysate. ICC/IF: HeLa cells, LLCPK1 (Sus scrofa kidney epithelial cell line). Flow Cytometry: HeLa cells. IP: HeLa whole cell extract.
  • General notes

    This product was changed from ascites to tissue culture supernatant on 17th September 2018. Please note that the dilutions may need to be adjusted accordingly. If you have any questions, please do not hesitate to contact our scientific support team.

    The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing this with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation. Please check that this product meets your needs before purchasing.

    If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, along with publications, customer reviews and Q&As

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Upon delivery aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
  • Storage buffer

    pH: 7.40
    Constituent: 100% PBS
  • Concentration information loading...
  • Purity

    Protein A purified
  • Purification notes

    Purified from TCS
  • Clonality

    Monoclonal
  • Clone number

    9G3
  • Myeloma

    unknown
  • Isotype

    IgG2a
  • Light chain type

    unknown
  • Research areas

    • Cell Biology
    • Cell Cycle
    • Cell Division
    • Spindle
    • Epigenetics and Nuclear Signaling
    • Cell cycle
    • Cell Cycle Inhibitors
    • Other
    • Cancer
    • Cell cycle
    • Cell division

Associated products

  • Compatible Secondaries

    • Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) (ab150113)
    • Goat Anti-Mouse IgG H&L (HRP) (ab205719)
    • Goat Anti-Mouse IgG H&L (DyLight® 488) preadsorbed (ab96879)
  • Isotype control

    • Mouse IgG2a, kappa monoclonal [MG2a-53] - Isotype control (ab18415)
  • Recombinant Protein

    • Recombinant Human HEC1/HEC protein (ab131907)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab3613 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
Flow Cyt
Use 1µg for 106 cells.

ab170191 - Mouse monoclonal IgG2a, is suitable for use as an isotype control with this antibody. 

IP
Use at an assay dependent concentration.

See image legend for details. 

WB (4)
1/500 - 1/3000. Detects a band of approximately 80 kDa (predicted molecular weight: 74 kDa).
ICC/IF (6)
1/100 - 1/1000.

See protocol in the legend for the HeLa cell image. 

Notes
Flow Cyt
Use 1µg for 106 cells.

ab170191 - Mouse monoclonal IgG2a, is suitable for use as an isotype control with this antibody. 

IP
Use at an assay dependent concentration.

See image legend for details. 

WB
1/500 - 1/3000. Detects a band of approximately 80 kDa (predicted molecular weight: 74 kDa).
ICC/IF
1/100 - 1/1000.

See protocol in the legend for the HeLa cell image. 

Target

  • Function

    Acts as a component of the essential kinetochore-associated NDC80 complex, which is required for chromosome segregation and spindle checkpoint activity. Required for kinetochore integrity and the organization of stable microtubule binding sites in the outer plate of the kinetochore.
  • Sequence similarities

    Belongs to the NDC80/HEC1 family.
  • Developmental stage

    Expression peaks in mitosis.
  • Post-translational
    modifications

    Phosphorylation begins in S phase of the cell cycle and peaks in mitosis. Phosphorylated by NEK2. May also be phosphorylated by AURKA and AURKB.
  • Cellular localization

    Nucleus. Chromosome > centromere > kinetochore. Localizes to kinetochores from late prophase to anaphase. Localizes specifically to the outer plate of the kinetochore.
  • Target information above from: UniProt accession O14777 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 10403 Human
    • Omim: 607272 Human
    • SwissProt: O14777 Human
    • Unigene: 414407 Human
    • Alternative names

      • from Entrez Gene ; antibody
      • HEC antibody
      • Highly expressed in cancer antibody
      • Highly expressed in cancer protein antibody
      • Highly expressed in cancer rich in leucine heptad repeats antibody
      • HsHec1 antibody
      • hsNDC80 antibody
      • Kinetochore associated 2 antibody
      • Kinetochore associated protein 2 antibody
      • Kinetochore protein Hec1 antibody
      • Kinetochore protein NDC80 homolog antibody
      • Kinetochore-associated protein 2 antibody
      • KNTC2 antibody
      • ndc80 antibody
      • NDC80 homolog kinetochore complex component antibody
      • NDC80 kinetochore complex component homolog antibody
      • NDC80, S. cerevisiae, homolog of antibody
      • NDC80_HUMAN antibody
      • Retinoblastoma associated protein HEC antibody
      • Retinoblastoma-associated protein HEC antibody
      • TID3 antibody
      see all

    Images

    • Immunocytochemistry/ Immunofluorescence - Anti-HEC1/HEC antibody [9G3] (ab3613)
      Immunocytochemistry/ Immunofluorescence - Anti-HEC1/HEC antibody [9G3] (ab3613)

      Immunocytochemistry/ Immunofluorescence analysis of HeLa cells labeling HEC1/HEC at the kinetochore with ab3613 at 1/500 (green). Cells were fixed in 4% paraformaldehyde at room temperature for 15 minutes. The cells were permeabilized with 0.1% Triton X-100 in PBS at room temperature for 4 minutes. The cells were blocked in 2.5% BSA/PBS at room temperature for 30 minutes. ab3613 was incubated at 4°C overnight. The secondary antibody was a Rabbit IgG antibody (Alexa Fluor 488), 1/2000 (keep from light), room temperature for 1hour. Washing with PBS 3 x 3 minutes. Red: alpha Tubulin 4a, a cytoskeleton marker, stained by an alpha Tubulin 4a antibody.
      Blue: Hoechst 33342 staining. 

      Synchronized condition: Suggest to treat cells with Nocodazole (10ng/ml, 24hr).

    • Western blot - Anti-HEC1/HEC antibody [9G3] (ab3613)
      Western blot - Anti-HEC1/HEC antibody [9G3] (ab3613)
      All lanes : Anti-HEC1/HEC antibody [9G3] (ab3613) at 1/1000 dilution

      Lane 1 : 293T whole cell lysate
      Lane 2 : A431 whole cell lysate
      Lane 3 : HepG2 whole cell lysate
      Lane 4 : HeLa whole cell lysate
      Lane 5 : HeLa nuclear extract

      Lysates/proteins at 30 µg per lane.

      Secondary
      All lanes : Mouse IgG antibody (HRP) at 1/5000 dilution

      Developed using the ECL technique.

      Predicted band size: 74 kDa



      7.5% gel.

      Running condition: 80V, 15min; 140V, 40min.

      Transfer condition: Semi-dry, 18 V, 60min (Nitrocellulose membrane).

      Blocking condition: 5% non-fat milk in TBST, RT, 60min.

      Primary antibody incubation: 4°C overnight.

      Secondary antibody incubation: Room temperature for 1 hour.

      Washing condition: 5 ml TBST, 4 x 5min.

      ECL detection. 

    • Immunocytochemistry/ Immunofluorescence - Anti-HEC1/HEC antibody [9G3] (ab3613)
      Immunocytochemistry/ Immunofluorescence - Anti-HEC1/HEC antibody [9G3] (ab3613)This image is courtesy of an anonymous Abreview

      ab3613 at 1/1000 dilution staining HeLa cells by ICC/IF. The cells were formaldehyde fixed and blocked with 5% BSA prior to incubation with the antibody for 2 hours. An Alexa-Fluor ® 488 conjugated goat anti-mouse antibody was used as the secondary.

      See Abreview

    • Immunoprecipitation - Anti-HEC1/HEC antibody [9G3] (ab3613)
      Immunoprecipitation - Anti-HEC1/HEC antibody [9G3] (ab3613)

      HEC1/HEC was immunoprecipitated using 0.5mg Hela whole cell extract, 10µg of Mouse monoclonal to HEC1/HEC and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
      The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
      Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab3613.
      Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1/5000 dilution.
      Band: Band: 76kDa: HEC1/HEC.

    • Immunocytochemistry/ Immunofluorescence - Anti-HEC1/HEC antibody [9G3] (ab3613)
      Immunocytochemistry/ Immunofluorescence - Anti-HEC1/HEC antibody [9G3] (ab3613)This image is courtesy of Scott Slattery and Mike Mancini

      HeLa cells were stained with anti-HEC1/HEC (ab2613; in green) and DAPI (blue) in panel 1, and with anti-HEC1/HEC (green) and SH-CREST (red) to stain the centromeres in panel 2. Fix the cells 30 minutes on ice in 4% formaldehyde in PEM. Quench autofluorescence 2 x 5 min. with 1 mg/ml Na borohydride or 100 mM ammonium chloride in PEM. Permeablize 30 min. with 0.5% TX-100 in PEM. Block 30 minutes in 5% milk in TBST. Primary antibody incubated at 1/1000 overnight at 4oC diluted in 5% milk in TBST. Secondary antibody 1 hour at RT diluted in 5% milk in TBST. Post-fix 20 min. on ice in 4% formaldehyde in PEM. Quench autofluorescence 2 x 5 min. with ammonium chloride in PEM. Counterstain with DAPI in TBST. Mount with ProLong Gold antifade reagent from Invitrogen. Notes: Ample washing between each step. TBST = Tris buffered saline + 0.1% Tween. PEM = 80 mM K-PIPES, pH 6.8, 5 mM EGTA, 2 mM MgCl2.

    • Western blot - Anti-HEC1/HEC antibody [9G3] (ab3613)
      Western blot - Anti-HEC1/HEC antibody [9G3] (ab3613)
      Anti-HEC1/HEC antibody [9G3] (ab3613) at 1/1000 dilution (4? , O/N) + HeLa cell lysate at 30 µg

      Secondary
      Mouse IgG antibody (HRP) at room temperature for 1 hour at 1/5000 dilution

      Predicted band size: 74 kDa



      Running conditions: run at 80V for 15 minutes then at 140V for 40 minutes

      Transfer conditions: Semi-dry at 18 V for 60min (NC membrane)

      Blocking conditions: 5% non-fat milk in TBST, RT, 60min.

      Washing conditions: 5 ml TBST, 4 x 5min

      Exposure system used: Trident plus Western HRP Substrate

    • Flow Cytometry - Anti-HEC1/HEC antibody [9G3] (ab3613)
      Flow Cytometry - Anti-HEC1/HEC antibody [9G3] (ab3613)
      Overlay histogram showing HeLa cells stained with ab3613 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab3613, 1µg/1x106 cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HeLa cells fixed with 80% methanol (5 min) /permeabilized in 0.1% PBS-Tween used under the same conditions.

    • Immunocytochemistry/ Immunofluorescence - Anti-HEC1/HEC antibody [9G3] (ab3613)
      Immunocytochemistry/ Immunofluorescence - Anti-HEC1/HEC antibody [9G3] (ab3613)

      Anti-HEC1/HEC antibody (ab3613) labels the kinetochores of mitotic cells in LLCPK1 (Sus scrofa kidney epithelial cell line) cell lines. Merge shows an overlay of DNA (stained with DAPI, red) and HEC1/HEC (green).
      This image was kindly supplied as part of the review submitted by Marko Kallio.

    Protocols

    • Flow cytometry protocols
    • Immunoprecipitation protocols
    • Immunocytochemistry & immunofluorescence protocols
    • Western blot protocols

    Click here to view the general protocols

    Datasheets and documents

    • Datasheet download

      Download

    References (102)

    Publishing research using ab3613? Please let us know so that we can cite the reference in this datasheet.

    ab3613 has been referenced in 102 publications.

    • Roscioli E  et al. Ensemble-Level Organization of Human Kinetochores and Evidence for Distinct Tension and Attachment Sensors. Cell Rep 31:107535 (2020). PubMed: 32348762
    • Barroso-Vilares M  et al. Small-molecule inhibition of aging-associated chromosomal instability delays cellular senescence. EMBO Rep 21:e49248 (2020). PubMed: 32134180
    • Martins NMC  et al. H3K9me3 maintenance on a human artificial chromosome is required for segregation but not centromere epigenetic memory. J Cell Sci 133:N/A (2020). PubMed: 32576667
    • Renda F  et al. Effects of malleable kinetochore morphology on measurements of intrakinetochore tension. Open Biol 10:200101 (2020). PubMed: 32634373
    • Zhang Y  et al. Biallelic loss of function NEK3 mutations deacetylate a-tubulin and downregulate NUP205 that predispose individuals to cilia-related abnormal cardiac left-right patterning. Cell Death Dis 11:1005 (2020). PubMed: 33230144
    View all Publications for this product

    Customer reviews and Q&As

    Show All Reviews Q&A
    Submit a review Submit a question

    1-10 of 18 Abreviews or Q&A

    Immunocytochemistry/ Immunofluorescence abreview for Anti-HEC1/HEC antibody [9G3]

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (HeLa)
    Permeabilization
    Yes - PBS/0.2% Triton X-100 for 30 minutes
    Specification
    HeLa
    Blocking step
    Serum as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 23°C
    Fixative
    Formaldehyde
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Weiguo Zhang

    Verified customer

    Submitted Aug 07 2015

    Immunocytochemistry/ Immunofluorescence abreview for Anti-HEC1/HEC antibody [9G3]

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (HEK293)
    Permeabilization
    Yes - triton
    Specification
    HEK293
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
    Fixative
    Paraformaldehyde
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Abcam user community

    Verified customer

    Submitted Jun 20 2014

    Immunocytochemistry/ Immunofluorescence abreview for Anti-HEC1/HEC antibody [9G3]

    Good
    Abreviews
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Hamster Cell (Chinese Hamster Ovary, T-REx™-CHO Cell Line)
    Permeabilization
    Yes - Triton 1%
    Specification
    Chinese Hamster Ovary, T-REx™-CHO Cell Line
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 37°C
    Fixative
    Paraformaldehyde
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    DR. Eleni Petsalaki

    Verified customer

    Submitted Oct 16 2013

    Immunocytochemistry/ Immunofluorescence abreview for Anti-HEC1/HEC antibody [9G3]

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (Colon carcinoma)
    Permeabilization
    Yes - 0.5% Triton in PHEM
    Specification
    Colon carcinoma
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 37°C
    Fixative
    Paraformaldehyde
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    DR. Eleni Petsalaki

    Verified customer

    Submitted Oct 08 2012

    Immunohistochemistry (Frozen sections) abreview for Anti-HEC1/HEC antibody [9G3]

    Poor
    Abreviews
    Abreviews
    abreview image
    Application
    Immunohistochemistry (Frozen sections)
    Sample
    Mouse Tissue sections (Brain (Embryonic day 17.5))
    Permeabilization
    Yes - 0.5% Triton
    Specification
    Brain (Embryonic day 17.5)
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 4% · Temperature: 23°C
    Fixative
    Paraformaldehyde
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Abcam user community

    Verified customer

    Submitted Aug 07 2012

    Question

    Sure. The ratio I used for WB was 1:1000, and the antibody and membrane were incubated in 5% milk for overnight. Following the incubation, the membrane was washed by 0.05% TBS-T buffer for three times (every 10 minutes), and then incubated with secondary antibody for 1 hour, and the secondary antibody was washed out for three times also after the incubation. Attached picture is the film was exposed for 5 seconds, the area with black is the membrane incubated with Hec1 antibody. Let me know if more information is needed, thanks.

    Read More

    Abcam community

    Verified customer

    Asked on Jan 25 2013

    Answer

    In general, issues like this arise from the secondary antibody. Often the antibody is too concentrated or is reacting with something in you buffers (usually milk or BSA) Have you had the opportunity to reduce the concentration of the secondary antibody when using this product or have you tried changing your blocking/incubation buffer? You may sometimes see these types of problem before film exposure by looking at the membrane in a darkroom after ECL. If the membrane is glowing then you should allow it to 'cool down' for up to 1 hour before exposure to film. I was also wondering if you could let me know what type of ECL reagent you are using? A high intensity ECL reagent like the Pierce Pico or Dura, GE Prime or Abcam Optiblot Ultradetect may be too sensitive for use with this product. If using a high sensitivity product we suggest switching to a lower sensitivity ECL reagent.

    Read More

    Abcam Scientific Support

    Answered on Jan 25 2013

    Question

    Hi,
    The Customer want to use this against Yeast . It is not tested and I did not
    find any . Can you please check ? Ekse it is ok if she get for protein NDC80
    also .
    Application : IF
    Host Mouse
    Reactivity : Yeast
    Regards

    Read More

    Abcam community

    Verified customer

    Asked on May 18 2012

    Answer

    Thank you for enquiry and your interest.

    I can confirm that this antibody (ab3613) recognizes Mouse, Human, Pig, African Green Monkey - it has not been tested in yeast and currently we do not have antibodies which detect HEC1 in Saccharomyces.

    If you need any further assistance in the future, please do not hesitate to contact me.

    Read More

    Abcam Scientific Support

    Answered on May 18 2012

    Question

    My note below was met with a request for more information regarding fixation conditions, etc. before a replacement was offered. We are loyal Abcam customers and want this taken care of as soon as possible. Please find details below: We tried IF again using this lot of HEC1 antibody, with similar results as before. We don't see kinetochore staining as we saw with a previous lot. The IF protocol failed using both methanol and formaldehyde fixation. The other channels and dapi looked fine. Furthermore, I didn't see specific Hec1 bands for my last western blot with your antibody either. Please send a replacement (9G3 HEC1 antibody) ASAP.

    Read More

    Abcam community

    Verified customer

    Asked on Feb 20 2012

    Answer

    I appreciate your cooperation and understand your concerns. It is regrettable that this product has not matched the standards of those previously.

    Under our Abpromise, we guarantee that our product will work in a the manner stated on the datasheet, e.g. Western Blot on porcine tissue. If the product has failed to do so, we offer scientific support, replace or refund of the product. Our Abpromise covers the product for 6 months.

    The details requested are nessesary in order for us to determine whether this product is covered by our Abpromise. If you could provide some information, such as species type or protocol conditions this would be very helpful. We appreciate your time taken to supply us this information. This information will also be helpful to our quality monitoring.

    I will be pleased to provide a free of charge replacmenet or credit note if the antibody is not working as it should be and has been purchased within the last 6 months. In order to process this I will need the Abcam order confirmation number or the PO number used to purchase the product.

    Thank you for your time. I look forward to hearing from you with the requested information and hope we can resolve this case as soon as possible.

    Read More

    Abcam Scientific Support

    Answered on Feb 20 2012

    Question

    To whom it may concern, We have been generally happy with the 9G3 HEC1 mouse monoclonal antibody for IF and western. However, we are having extremely poor results with the newest aliquot you sent. In an IF experiment repeated twice were other channels showed up an experienced researcher in our lab had no signal for HEC1 - this is very disappointing. Other channels on the same slide looked perfect. The offending order is listed below. The batch ordered 12/15/2011 has not worked, the batch from 2010 worked beautifully. HEC1 antibody [9G3] (ab3613) Will you please send us a replacement antibody from a different lot, same product? if we need to chat on the phone about this just give me a call - thanks! besides this latest issue we are huge abcam supporters

    Read More

    Abcam community

    Verified customer

    Asked on Jan 25 2012

    Answer

    Thank you for contacting Abcam.

    I have called and left a message with you in regards to this issue. Feel free to disregard that call should this be a better forum for communicating.

    I am sorry that the latest lot of this product is not performing well for you. Before I may issue a replacement however I was hoping that you could please send me the protocol that you have used, any troubleshooting steps that you might have performed and any images should you have them. Please be as specific as possible as this data will help us pinpoint issues that the antibody might have and test them as necessary.

    Please let me know if you have any questions.

    Read More

    Abcam Scientific Support

    Answered on Jan 25 2012

    Question

    Customer kindly asked how the antibody should be diluted at 1/1000 dilution.

    Read More

    Abcam community

    Verified customer

    Asked on Jan 04 2012

    Answer

    Thank you very much for your phone call. The dilution given on the datasheet is for guideline purpose only. The dilutions have to be optimized by the end user based on cells or tissue sections they use. We recommend using a range e.g. 1/500, 1/1000 and 1/2000 dilution; this range will helps to get best optimized results at particular dilutions. 1/1000 dilution =1 ul of antibody diluted in 999 ul of buffer (1-5%BSA + PBS). or 10 ul of antibody solution in 9990 ul of buffer will give the same 1/1000 dilution with total volume of 10ml. We recommend aliquoting 10 ul per vial and storing the antibody neat at -20C or -80C (as recommended on datasheet). Antibodies can also be diluted in PBS with 1-3% BSAin casethere is need for diluted antibody to be stored. I hope this info will be helpful. SHould you have any other inquiry please do not hesitate to contact us.

    Read More

    Abcam Scientific Support

    Answered on Jan 04 2012

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