Overview

  • Product name
    Anti-ORC4L antibody - ChIP Grade
    See all ORC4L primary antibodies
  • Description
    Goat polyclonal to ORC4L - ChIP Grade
  • Host species
    Goat
  • Tested applications
    Suitable for: ChIP, WB, ELISAmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
    Predicted to work with: Cow, Dog
  • Immunogen

    Synthetic peptide: DVRQWATSSLSWL, corresponding to C terminal amino acids 424-436 of Human ORC4L.

  • Positive control
    • Jurkat and NSO (mouse) cell lysates.
  • General notes
    Official Gene Symbol - ORC4L GenBank Accession Number – NP_002543 LocusLink ID - 5000

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • Storage buffer
    Preservative: 0.02% Sodium Azide
    Constituents: 0.5% BSA, Tris buffered saline, pH 7.3
  • Concentration information loading...
  • Purity
    Immunogen affinity purified
  • Purification notes
    Purified from goat serum by ammonium sulphate precipitation followed by antigen affinity chromatography using the immunizing peptide.
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab9641 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
ChIP Use at an assay dependent concentration.
WB Use a concentration of 0.5 - 2 µg/ml. Detects a band of approximately 50 kDa (predicted molecular weight: 50 kDa).Can be blocked with Human ORC4L peptide (ab23156).
ELISA Use at an assay dependent concentration.

Target

  • Function
    Component of the origin recognition complex (ORC) that binds origins of replication. DNA-binding is ATP-dependent, however specific DNA sequences that define origins of replication have not been identified so far. ORC is required to assemble the pre-replication complex necessary to initiate DNA replication.
  • Involvement in disease
    Defects in ORC4 are the cause of Meier-Gorlin syndrome type 2 (MGORS2) [MIM:613800]. MGORS2 is a syndrome characterized by bilateral microtia, aplasia/hypoplasia of the patellae, and severe intrauterine and postnatal growth retardation with short stature and poor weight gain. Additional clinical findings include anomalies of cranial sutures, microcephaly, apparently low-set and simple ears, microstomia, full lips, highly arched or cleft palate, micrognathia, genitourinary tract anomalies, and various skeletal anomalies. While almost all cases have primordial dwarfism with substantial prenatal and postnatal growth retardation, not all cases have microcephaly, and microtia and absent/hypoplastic patella are absent in some. Despite the presence of microcephaly, intellect is usually normal.
  • Sequence similarities
    Belongs to the ORC4 family.
  • Cellular localization
    Nucleus.
  • Information by UniProt
  • Database links
  • Alternative names
    • Origin recognition complex, subunit 4, S. cerevisiae, homolog of antibody
    • FLJ46668 antibody
    • HSORC4 antibody
    • ORC 4 antibody
    • ORC 4L antibody
    • ORC 4P antibody
    • ORC4 antibody
    • ORC4_HUMAN antibody
    • ORC4L antibody
    • ORC4L protein antibody
    • ORC4P antibody
    • Origin recognition complex subunit 4 (yeast homolog) like antibody
    • Origin recognition complex subunit 4 antibody
    • Origin recognition complex subunit 4 like (yeast) antibody
    • Origin recognition complex subunit 4 like antibody
    • origin recognition complex, subunit 4 homolog antibody
    • Origin recognition complex, subunit 4, S. cerevisiae, homolog-like antibody
    see all

Images

  • Anti-ORC4L antibody - ChIP Grade (ab9641) at 0.5 µg/ml + Mouse heart lysate at 35 µg

    Predicted band size: 50 kDa

  • Anti-ORC4L antibody - ChIP Grade (ab9641) at 0.5 µg/ml + Rat Heart lysate in RIPA buffer at 35 µg

    Developed using the ECL technique.

    Predicted band size: 50 kDa
    Observed band size: 50 kDa

  • ab9641 (2µg/ml) staining of Jurkat lysate (RIPA buffer, 1.4E5 cells per lane). Detected by western blot using chemiluminescence.
  • All lanes : Anti-ORC4L antibody - ChIP Grade (ab9641) at 1 µg/ml

    Lane 1 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
    Lane 2 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate
    Lane 3 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Rabbit polyclonal to Goat IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 50 kDa
    Observed band size: 49 kDa
    why is the actual band size different from the predicted?
    Additional bands at: 38 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 20 minutes

References

This product has been referenced in:
  • Hossain M & Stillman B Opposing roles for DNA replication initiator proteins ORC1 and CDC6 in control of Cyclin E gene transcription. Elife 5:N/A (2016). IP ; Human . Read more (PubMed: 27458800) »
  • Zhang R  et al. Targeting GLI by GANT61 involves mechanisms dependent on inhibition of both transcription and DNA licensing. Oncotarget 7:80190-80207 (2016). ICC/IF ; Human . Read more (PubMed: 27863397) »
See all 9 Publications for this product

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Question

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER 202754 DESCRIPTION OF THE PROBLEM no signal SAMPLE a lymphoblastoid cell line nuclear extract PRIMARY ANTIBODY AbCam 9641, 20ul in 2ml sample, 4C, overnight DETECTION METHOD Sybr Green ANTIBODY STORAGE CONDITIONS aliquoted and stored at -20C SAMPLE PREPARATION Crosslink, then wash with PBS twice, incubate at cell lysate buffer (5mM PIPES, 85mM KCl, 25ul NP40, 1:100 Protease Inhibitor cocktail) for 10 min on ice, spin down, and resuspend in nuclear lysate buffer (50 mM Tris-HCl, ph8, 10mM EDTA, 1% SDS, 1:100 protease inhibitor cocktail), then sonicate X-CHIP or N-CHIP X-ChIP CROSSLINKING 1% formaldehyde, at 37C for 15 min DNA FRAGMENTATION sonication, size is enriched between 500bp to 2Kb as checked on a 1% agarose gel IP STEP 10 to 15 million cells in 200 ul nuclear lysate buffer, then dilute with 9 volume of ChIP dilution buffer (Upstate), add 160 ul Salmone Sperm DNA/Protein A Agarose Slurry, 4C, 30 min, rotation, then spin, save the supernantant, add in 20ul of antibody, 4C, rotation overnight, then add 120ul beads, 1hr at 4C, and then wash the beads with low salt, high salt, LiCl, and TE (Upstate), then elute with 1% SDS in 0.1M NaHCO3 we also try to conjugate beads and antibody first DECROSSLINKING 20 ul 5M NaCl was added to the sample (final concentation 0.2M), 65C for 4 hr DNA PURIFICATION Phenol Chloroform extraction PELLET AFTER PRECIPITATION EtOH precipitation PRIMERS TESTED ON GENOMIC/INPUT DNA Yes, on genomic DNA with Sybr Green, no primer dimer NO-TEMPLATE CONTROL IN PCR Yes water control HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? in the 2nd trial, we conjugate the beads with the antibody first

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Answer

Thank you for your enquiry. I am sorry to hear that you have been having difficulties with this antibody. This antibody was assigned "ChIP grade" status following its application in the following publication; Todorovic V et al. Human origins of DNA replication selected from a library of nascent DNA. Mol Cell 19:567-75 (2005). PubMed: 16109380 I have examined the protocol that they employed and the protocol that you are using is indeed very similar. As a control I would like to ask whether you have been incorporating a control antibody such as anti-histone H3. Also can you tell me the positive control loci that you have been using? Do you have a locus that you are expecting ORC4L to be recruited to? Is ORC4L constantly associated with this locus, or recruited in a stepwise process? I look forward to hearing from you.

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Answer

Thank you for your enquiry. Unfortunately, lot #35325 is no longer in stock. Please rest assured, however, that we guarantee that ab9641 will work as stated on the online datasheet or we will provide a replacement or refund. Please contact us again if you have any additional questions.

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Answer

Thank you for your e-mail. I'm really sorry to hear you are having problems with this antibody and would like to give you a few suggestions. You have not mentioned the amount of protein loaded onto the gel. If it is under 30-40ug this may be too low and hence there is very little ORC4L protein to be detected. You may also have a very low concentration of ORC4L in your samples and a western blot detection may not be enough to detect the protein. I would recommend trying a cell line which is known to express ORC4L at detectable levels, such as Jurkat and NSO (mouse) cell lysates (we have Jurkat cell lysat in our catalogue:ab7899). Can I confirm also that you add Tween 20 in the buffer in which the primary antibody is diluted in, as it helps the penetration of the antibody into the membrane? Do you block the membrane with milk or BSA? Finally, our datasheet recommends trying 1-3ug/ml so I would suggest you try 3ug/ml and maybe 5ug/ml too? This may help detecting any low amounts of protein you may have in your samples. I hope this information helps, but please do not hesitate to contact me if you need further advice, Good luck with your westerns!

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