Anti-Separase antibody [XJ11-1B12] (ab16170)

Overview

  • Product nameAnti-Separase antibody [XJ11-1B12]
    See all Separase primary antibodies
  • Description
    Mouse monoclonal [XJ11-1B12] to Separase
  • Tested applicationsSuitable for: Flow Cyt, WB, ICC/IFmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Maltose binding protein fusion protein, corresponding to C terminal amino acids 1866-1996 of Human Separase.

  • Positive control
    • This antibody gave a positive signal in MDA-MB-231 Whole Cell Lysate.
  • General notes


    Separase antibodies can be used as a specific marker for centrosomes of mitotic cells. The staining of separase in centrosomes can be detected from prophase of mitosis up until anaphase.

Properties

  • FormLiquid
  • Storage instructionsShipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.
  • Storage bufferpH: 7.40
    Preservative: 0.02% Sodium azide
    Constituent: PBS
  • Concentration information loading...
  • PurityIgG fraction
  • Primary antibody notesSeparase antibodies can be used as a specific marker for centrosomes of mitotic cells. The staining of separase in centrosomes can be detected from prophase of mitosis up until anaphase.
  • ClonalityMonoclonal
  • Clone numberXJ11-1B12
  • IsotypeIgG1
  • Light chain typekappa
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab16170 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. ab170190-Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
WB Use a concentration of 10 µg/ml. Detects a band of approximately 170 kDa (predicted molecular weight: 233 kDa).
ICC/IF Use at an assay dependent concentration. Used at a dilution of 1/100 for 1 hr on HeLa cells (see Abreview).

Target

  • FunctionCaspase-like protease, which plays a central role in the chromosome segregation by cleaving the SCC1/RAD21 subunit of the cohesin complex at the onset of anaphase. During most of the cell cycle, it is inactivated by different mechanisms.
  • Sequence similaritiesBelongs to the peptidase C50 family.
  • Post-translational
    modifications
    Autocleaves. This function, which is not essential for its protease activity, is unknown.
    Phosphorylated by CDK1. There are 8 Ser/Thr phosphorylation sites. Among them, Ser-1126 phosphorylation is the major site, which conducts to the enzyme inactivation.
  • Cellular localizationCytoplasm. Nucleus.
  • Information by UniProt
  • Database links
  • Alternative names
    • Caspase like protein ESPL1 antibody
    • Caspase-like protein ESPL1 antibody
    • ESP 1 antibody
    • ESP1 antibody
    • ESPL 1 antibody
    • Espl1 antibody
    • ESPL1_HUMAN antibody
    • Extra spindle poles like 1 antibody
    • Extra spindle poles like 1 protein antibody
    • Extra spindle poles-like 1 protein antibody
    • FLJ46492 antibody
    • KIAA0165 antibody
    • Separase antibody
    • Separin antibody
    • Similar to fission yeast cut1and gene antibody
    • SSE antibody
    see all

Anti-Separase antibody [XJ11-1B12] images

  • ab16170 stained MCF7 cells. The cells were 100% methanol fixed for 5 minutes at -20°C and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1hour at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab16170 at 10µg/ml) overnight at +4°C. The secondary antibody (pseudo-colored green) was Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed (ab150117) used at a 1/1000 dilution for 1hour at room temperature. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.

  • Overlay histogram showing HeLa cells stained with ab16170 (red line). The cells were fixed with 80% methanol (5 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 (ab16170, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

  • Anti-Separase antibody [XJ11-1B12] (ab16170) at 10 µg/ml + MDA-MB-231 (Human breast adenocarcinoma cell line) Whole Cell Lysate at 20 µg

    Secondary
    Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
    Developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 233 kDa
    Observed band size : 170 kDa (why is the actual band size different from the predicted?)
    Additional bands at : 100 kDa,70 kDa (possible cleavage fragment). We are unsure as to the identity of these extra bands.

    Exposure time : 3 minutes

References for Anti-Separase antibody [XJ11-1B12] (ab16170)

This product has been referenced in:
  • Agircan FG & Schiebel E Sensors at centrosomes reveal determinants of local separase activity. PLoS Genet 10:e1004672 (2014). Read more (PubMed: 25299182) »
  • Han X & Poon RY Critical differences between isoforms of securin reveal mechanisms of separase regulation. Mol Cell Biol 33:3400-15 (2013). Read more (PubMed: 23798554) »

See all 6 Publications for this product

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Human Cell lysate - whole cell (Hela)
Loading amount 12500 cells
Specification Hela
Treatment Transfected with siRNA duplexes (control or targetting separase), subsequent nocodazole arrest
Gel Running Conditions Reduced Denaturing (4-12% PAGE)
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
Username

Abcam user community

Verified customer

Submitted Sep 16 2010

Application Immunoprecipitation
Sample Human Cell lysate - whole cell (MDA-MB-231 breast cancer cell line)
Total protein in input 40 µg
Specification MDA-MB-231 breast cancer cell line
Immuno-precipitation step Protein G
Username

Abcam user community

Verified customer

Submitted Oct 10 2008

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Human Cell lysate - whole cell (MDA-MB-231 breast cancer cell line)
Loading amount 40 µg
Specification MDA-MB-231 breast cancer cell line
Gel Running Conditions Reduced Denaturing (gel 8 %)
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Oct 10 2008

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunocytochemistry/ Immunofluorescence
Sample Human Cell (HeLa)
Specification HeLa
Fixative Methanol
Blocking step Serum as blocking agent for 45 minute(s) · Concentration: 5%
Username

Dr. Ashraf Dallol

Verified customer

Submitted Jun 22 2006

Thank you for your enquiry. Here we are sending a general Western blot protocol. The information is only intended as a guide. The researchers should determine what protocol best meets their needs: Sample Preparation 1. Remove media and wash ...

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