Overview

  • Product name
  • Description
    Goat polyclonal to Metnase
  • Host species
    Goat
  • Tested applications
    Suitable for: WBmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Synthetic peptide corresponding to Human Metnase aa 658-671 (C terminal).
    Sequence:

    RWQKCVDCNGSYFD


    (Peptide available as ab22960)

  • Positive control
    • Daudi lysate.
  • General notes

    Principal Names – Metnase; SET domain and mariner transposase fusion gene. Official Gene Symbol - Metnase. GenBank Accession Number – NP_006506. LocusLink ID - 6419 (human). Gene Ontology terms - transposase activity

    Previously labelled as SETMAR. 

Properties

Applications

Our Abpromise guarantee covers the use of ab3823 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 Use a concentration of 1 - 3 µg/ml. Detects a band of approximately 70-75 kDa.Can be blocked with Human Metnase peptide (ab22960).

Approx 70 - 75 kDa band seen in Daudi lysate.

Target

  • Function
    Histone methyltransferase that methylates 'Lys-4' and 'Lys-36' of histone H3, 2 specific tags for epigenetic transcriptional activation. Specifically mediates dimethylation of H3 'Lys-36'. Has sequence-specific DNA-binding activity and recognizes the 19-mer core of the 5'-terminal inverted repeats (TIRs) of the Hsmar1 element. Has DNA nicking activity. Has in vivo end joining activity and may mediate genomic integration of foreign DNA.
  • Tissue specificity
    Widely expressed, with highest expression in placenta and ovary and lowest expression in skeletal muscle.
  • Sequence similarities
    In the N-terminal section; belongs to the histone-lysine methyltransferase family.
    In the C-terminal section; belongs to the mariner transposase family.
    Contains 1 post-SET domain.
    Contains 1 pre-SET domain.
    Contains 1 SET domain.
  • Domain
    The mariner transposase Hsmar1 region mediates DNA-binding. It has no transposase activity because the active site contains an Asn in position 610 instead of a Asp residue.
  • Cellular localization
    Nucleus. Chromosome.
  • Information by UniProt
  • Database links
  • Alternative names
    • Histone lysine N methyltransferase antibody
    • Histone lysine N methyltransferase SETMAR antibody
    • Hsmar 1 antibody
    • Hsmar1 antibody
    • Mariner transposase Hsmar1 antibody
    • Metnase antibody
    • SET domain and mariner transposase fusion antibody
    • SET domain and mariner transposase fusion gene antibody
    • SET domain and mariner transposase fusion gene containing protein antibody
    • SET domain and mariner transposase fusion gene-containing protein antibody
    • Setmar antibody
    • SETMR_HUMAN antibody
    see all

Images

  • ab3823 staining (2µg/ml) of Daudi lysate (RIPA buffer, 30µg total protein per lane).  Primary incubated for 12 hours.  Detected by western blot using chemiluminescence. ab3823 staining (2µg/ml) of Daudi lysate (RIPA buffer, 30µg total protein per lane). Primary incubated for 12 hours. Detected by western blot using chemiluminescence.

References

This product has been referenced in:
  • Van Duyne R  et al. Lysine methylation of HIV-1 Tat regulates transcriptional activity of the viral LTR. Retrovirology 5:40 (2008). WB ; Human . Read more (PubMed: 18498648) »
See 1 Publication for this product

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Question

Here is the information you requested. Lot #15383, Invoice # 12808 > 1. Please describe the problem (high background, wrong band size, more > bands, no band etc). In all Westerns using the primary antibody, multiple bands are present; in fact, when compared to a Coomassie stained gel, all bands visible in the Coomassie gel also appear to be stained in the Western blot. > > 2. On what material are you testing the antibody in WB? >Species? Human > Cell extract/ Nuclear extract? We have tested two independent cell extracts: an Imgenex Daudi lysate and an ATCC Daudi cell line we maintain in culture > Purified protein? No, crude extract > Recombinant protein? No > 3. How much protein did you load? 30 micrograms (according to Abcam protocol) > How did you prepare the lysate for the analysis (protease inhibitors etc)? The Imgenex Daudi cell lysate is ready-to-load, it comes with beta-mercaptoethanol and sample buffer added. The Daudi cell culture we maintain was prepared according to the Abcam protocol we received. >Did you heat the samples? No, not necessary for Imgenex cell lysate. Yes, for the Daudi cell culture we maintain (according to Abcam protocol) > > 4. Primary Antibody > Specification (in which species was it raised against)? Goat > At what dilution(s) have you tested this antibody? 0.5ug/ml, 1.0ug/ml, and 2ug/ml (2ug/ml is recommended in Abcam protocol) > Incubation time, wash step? Tested incubation time at both 1hr and overnight. Wash in 1X PBST (Tween tested at both 0.1% and 0.05%) 3X for 5-15min > > 5. Secondary Antibody > Specification (in which species was it raised against)? Rabbit anti-goat IgG purchased from Sigma (A-8919) > At what dilution(s) have you tested this antibody? 1:3000 and 1:4000 > Incubation time, wash step? 1hr, same washes tested as for primary antibody > Do you know whether the problems you are experiencing come from the > secondary? We have performed a "no primary" control, and there is almost no staining present. > > 6. What detection method are you using? HRP with DAB, metal-enhanced, developed 5-10 min. > > 7. Background bands > Have you eliminated the possibility that any background bands could > be due to the secondary antibody? (Run a â??No primaryâ?? control) Yes Is the blocking step sufficient? (We recommend blocking the membrane by > adding 20 ml of blocking buffer (5% non-fat dry milk, 0.1% Tween-20 in > TBS). Incubate for 2 h at room temperature or overnight at 4°C with > agitation) We use 5% non-fat dry milk, 0.1% Tween-20 in PBS overnight at 4C. Are your washing steps sufficiently stringent? (Multiple > short washes are more effective than fewer longer wash steps) We have washed 3X for 15 minutes each. >At what size are the bands migrating? Could they be degradation products of your target? It appears that all bands in the lysate are stained in the Western. >Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient) The attached image shows 30ug and 20ug of Imgenex Daudi lysate using the SETMAR primary antibody. The far left lane is our ladder, which doesn't hold up too well during the processing, but we marked the approximate positions of 39kD and 78kD. The SETMAR primary antibody should identify a 75kD protein. The far right lane is our positive control which is described in detail below. > 8. Optimization attempts How many times have you tried the Western? Seven. Do you obtain the same results every time e.g. are background bands > always in the same place? Yes What steps have you altered? We have altered primary antibody concentrations, secondary antibody concentrations, incubation times, number of washes and length of time, Tween concentration, and lysate mass loaded. > > 9. Did you apply positive and negative controls along with the samples? > Please specify. Our positive control is alpha-actinin purified protein from chicken gizzard (Sigma A9776) detected with monoclonal anti-alpha-actinin from mouse (Sigma A5044), using an anti-mouse secondary antibody (Sigma) A9917.

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Answer

Thank you very much for your patience. The only suggestion that I have at this point (you have done pretty much everything!) is to try blocking with 3% BSA instead. The originator didn't see high background with this antibody and used a more sensitive detection system (ECL plus). Did you buy the antibody directly from us or from a distributor, such as Novus Biologicals? And what was the date? I'm not coming up with a record of this antibody being bought from your school. Thanks...

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Answer

The samples (in 1.5ml microtubes) were simply put into boiling water. So I would expect the temperature to be almost exactly 100 degrees centigrade.

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Answer

As stated on the datasheet we used the following conditions to get our result: ab3823 staining (2µg/ml) of Daudi lysate (RIPA buffer, 30µg total protein per lane). Primary incubated for 12 hours. Detected by western blot using chemiluminescence However additional information about our protocol is given below: - Lysis. Cell pellets were washed with ice-cold PBS. 1 ml of RIPA buffer was added per 1E8 cells and incubated on ice for 20 min, vortexing 2-3 times, briefly. The lysate was aliquotted into 1.5 ml microfuge tubes and centrifuged at 13,000 rpm for 5 min in a microfuge. The supernatant was transferred into clean tubes and its protein concentration was measured with BioRad protein assay. The concentration was then adjusted to 5 mg/ml with RIPA lysis buffer. An equal volume of 2 x SDS sample buffer was then added and the cell lysate was boiled for 5 minutes. Lysates were stored at -80C until use.(RIPA buffer = 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM PMSF, 1 mM EDTA, 5 µg/ml Aprotinin, 5 µg/ml Leupeptin, 1% Triton -100, 1% Sodium deoxycholate, 0.1% SDS). - SDS PAGE. Samples were run at 200V constant on a 12% acrylamide SDS-PAGE mini gel - using Biorad Mini-Protean 3 kit and protocols. Before loading samples had 5% (v/v) 2-ME added and were boiled for 3 minutes. - Transfer. We used a Biorad Mini Trans-Blot, constant 100 V for 1 hour. Transfer Buffer was 20 mM Tris pH 8.0, 150 mM Glycine, 10% Methanol. We transferred to Millipore PVDF membrane and stained with Ponceau Red to evaluate the transfer. - Staining. The membrane was blocked in 2.5% skimmed milk in TBS-T (TBS + 0.05% Tween-20) for 1 hr at room temperature with agitation. Primary antibody was incubated for 1 hr at room temperature with agitation. We used anti-goat-HRP at 1:3000 for 1 hr at room temperature with agitation. We washed with TBST three times after primary and secondary antibody, each wash lasting for 5-10 mins. ECL-plus (Amersham) was used rather than ECL, which is considerably more sensitive. Final detection was on autoradiography film.

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