Overview

  • Product name
    Anti-Separase antibody [XJ11-1B12]
    See all Separase primary antibodies
  • Description
    Mouse monoclonal [XJ11-1B12] to Separase
  • Host species
    Mouse
  • Tested applications
    Suitable 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

    This antibody clone is manufactured by Abcam.


    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.

    If you require this antibody in a particular buffer formulation or a particular conjugate for your experiments, please contact orders@abcam.com or you can find further information here.

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.
  • Storage buffer
    pH: 7.40
    Preservative: 0.02% Sodium azide
    Constituent: PBS
  • Concentration information loading...
  • Purity
    IgG fraction
  • Primary antibody 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.
  • Clonality
    Monoclonal
  • Clone number
    XJ11-1B12
  • Isotype
    IgG1
  • Light chain type
    kappa
  • 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

  • Function
    Caspase-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 similarities
    Belongs 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 localization
    Cytoplasm. 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

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

This product has been referenced in:
  • Karki M  et al. Precocious centriole disengagement and centrosome fragmentation induced by mitotic delay. Nat Commun 8:15803 (2017). WB ; Human . Read more (PubMed: 28607478) »
  • Seo MY  et al. Integrity of the Pericentriolar Material Is Essential for Maintaining Centriole Association during M Phase. PLoS One 10:e0138905 (2015). WB . Read more (PubMed: 26407333) »
See all 10 Publications for this product

Customer reviews and Q&As

1-6 of 6 Abreviews or Q&A

Answer

Thank you for calling us and for alerting us to the problem you are experiencing with our product. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly. If you wouldn't mind, could you please fill in the questionnaire with details of the experiments where ab16170 was used. This information will also allow us to investigate this case internally and initiate additional testing where necessary. If the product was purchased in the last six months and is being used according to our Abpromise, we would be happy to replace or refund the antibody. In regards to the reactivity of this antibody with chicken, unfortunately having compared the human (SwissProt Q14674) separase to that of chicken (NCBI XP_423289.2) the alignment is very poor and I would therefore not expect this antibody to detect the chicken protein. All the other antibodies we have for this target are also raised against the human protein and I can therefore not recommend an alternative. You may have more luck by searching using biocompare: www.biocompare.com If you have any further questions please do not hesitate to ask.

Read More
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

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

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

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%

Dr. Ashraf Dallol

Verified customer

Submitted Jun 22 2006

Answer

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 cells with sterile PBS. 2. Scrape cells into PBS and pellet in a centrifuge. 3. Resuspend pellet in 300 ul of RIPA buffer. 4. Pass lysate through 21 gauge needle 3-4 time to shear the DNA. 5. Incubate on ice for 30-60 min. 6. Spin at 15,000g for 20 minutes at 4ºC. 7. Remove and keep the supernatant. 8. Measure protein content in supernatant. 9. Add loading buffer to supernatant (50 ml/200 ml supernatant). 10. Add mercaptoethanol (5 ml/100 ml). 11. Boil samples for 5-10 minutes in heating block. II Sample Running and Transfer: 1. Follow recommendations of manufacture. 2. Verify transfer by staining with Ponceau S dye. 3. Mark blot with pencile III Detection: 1. Block blot in 5% milk/TBS/Tween in the container, 1hr, RT, on shaker; OR o/n in 4C on a rocker. 2. Dilute primary antibody in 5% milk/TBS and incubate with blot for 1hr RT, or o/n in cold room on a rocker. 3. Wash: 3 x 5 minutes in TBS/Tween. 4. Dilute secondary antibody in 5% milk/TBS, incubate 1hr at RT, on shaker. 5. Wash: 3 x 10 minutes in TBS/Tween. 6. Cut a pc. of transparency into 2 smaller pcs. that fit into the cassette; mix oxidizing and luminol reagent 1: 1, transfer onto a piece of parafilm, lay the blot over the solution for 1 min; then insert the blot between the 2 transparencies. Make sure to remove all bubbles. 7. Initially expose for 1 min and then adjust for the later trial. The signals will decay over time. The positive controls used for this experiment were 15 ug of non-induced 5C6 and induced 5C6 whole cell lysates. Both lysates gave a clear positive signal. The induced form gave a stronger signal, but the non-induced form was still very clear. We hope that this helps.

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