Overview

  • Product name
    Anti-ASK1 antibody [EP553Y]
    See all ASK1 primary antibodies
  • Description
    Rabbit monoclonal [EP553Y] to ASK1
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, IHC-P, ICC/IF, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Human
  • Immunogen

    Synthetic peptide corresponding to Human ASK1 aa 1-100 (N terminal).
    (Peptide available as ab224650)

  • Positive control
    • Human lung carcinoma tisuue, HeLa cell lysate, HeLa cells, RAW 264.7 whole cell lysates, Human pancreas, Mouse kidney.
  • General notes

    Rat: We have preliminary internal testing data to indicate this antibody may not react with this species. Please contact us for more information.

     

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab45178 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 1/1000. Detects a band of approximately 155 kDa (predicted molecular weight: 155 kDa).Can be blocked with ASK1 peptide (ab224650).

For unpurified use at 1/2000

IHC-P 1/50. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

ICC/IF 1/100 - 1/250.
Flow Cyt 1/100.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

Target

  • Function
    Component of a protein kinase signal transduction cascade. Phosphorylates and activates MAP2K4 and MAP2K6, which in turn activate the JNK and p38 MAP kinases, respectively. Overexpression induces apoptotic cell death.
  • Tissue specificity
    Abundantly expressed in heart and pancreas.
  • Sequence similarities
    Belongs to the protein kinase superfamily. STE Ser/Thr protein kinase family. MAP kinase kinase kinase subfamily.
    Contains 1 protein kinase domain.
  • Post-translational
    modifications
    Dephosphorylated and activated by PGAM5.
  • Information by UniProt
  • Database links
  • Alternative names
    • Apoptosis signal regulating kinase 1 antibody
    • Apoptosis signal-regulating kinase 1 antibody
    • ASK 1 antibody
    • ASK-1 antibody
    • ASK1 antibody
    • M3K5 antibody
    • M3K5_HUMAN antibody
    • MAP/ERK kinase kinase 5 antibody
    • MAP3K5 antibody
    • MAPK/ERK kinase kinase 5 antibody
    • MAPKKK5 antibody
    • MEK kinase 5 antibody
    • MEKK 5 antibody
    • MEKK5 antibody
    • Mitogen activated protein kinase kinase kinase 5 antibody
    • Mitogen-activated protein kinase kinase kinase 5 antibody
    see all

Images

  • Lane 1: Wild-type HAP1 cell lysate (20 µg)
    Lane 2: ASK1 knockout HAP1 cell lysate (20 µg)
    Lane 3: HeLa cell lysate (20 µg)
    Lane 4: A549 cell lysate (20 µg)
    Lanes 1 - 4: Merged signal (red and green). Green - ab45178 observed at 155, 160 kDa. Red - loading control, ab1240, observed at 124 kDa.

    Unpurified ab45178 was shown to recognize ASK1 when ASK1 knockout samples were used, along with additional cross-reactive bands. Wild-type and ASK1 knockout samples were subjected to SDS-PAGE. ab45178 and ab1240 (loading control to Vinculin) were diluted to 1/2000 and 1/10000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

  • Immunocytochemistry/ Immunofluorescence analysis of RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) cells labeling ASK1 with Purified ab45178 at 1:100 (9.9 µg/ml). Cells were fixed in 100% Methanol. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1:200 (2.5 �g/ml). Goat anti rabbit IgG (Alexa Fluor� 488, ab150077) was used as the secondary antibody at 1:1000 (2 �g/ml) dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.
  • Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling ASK1 with purified ab45178 at 1:100 dilution (10 µg/ml) (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor® 488) secondary antibody was used at 1:2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).
  • Immunofluorescent staining of HeLa cells using unpurified ab45178 (1/10).

  • Overlay histogram showing HeLa cells stained with unpurified ab45178 (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 (ab45178, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was a goat anti-rabbit DyLight® 488 (IgG H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

  • Unpurified ab45178 (1/10) staining human ASK1 in human lung carcinoma by immunohistochemistry using paraffin embedded tissue.

  • Anti-ASK1 antibody [EP553Y] (ab45178) at 1/2000 dilution (Unpurified) + HeLa cell lysate at 10 µg

    Secondary
    Goat anti-rabbit, HRP labelled at 1/2000 dilution

    Predicted band size: 155 kDa
    Observed band size: 155 kDa

References

This product has been referenced in:
  • Giuliano CJ  et al. MELK expression correlates with tumor mitotic activity but is not required for cancer growth. Elife 7:N/A (2018). Read more (PubMed: 29417930) »
  • Cheng R  et al. ß-TrCP-dependent degradation of ASK1 suppresses the induction of the apoptotic response by oxidative stress. Biochim Biophys Acta Gen Subj 1862:2271-2280 (2018). Read more (PubMed: 30031111) »
See all 15 Publications for this product

Customer reviews and Q&As

1-9 of 9 Abreviews or Q&A

Application
Western blot
Loading amount
25 µg
Gel Running Conditions
Reduced Denaturing
Sample
Mouse Cell lysate - whole cell (LIVER)
Specification
LIVER
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 Nov 10 2014

Answer

Currently we do not offer ab45178 without BSA, however we may be able to make this custom formulation for you. I'll put you in touch with our RabMAb Technical Account Executive and he can help you look into this further.

Read More

Answer

The concentration of ab45178, lot GR48990-3, is 1.262 mg/mL.

I have CC's your request for a quote for 10 vials of this lot to our bulk order specialist, who can be reached at sales@abcam.com. She will be able to provide you a quote which includes a discount for purchasing at least 10 vials.

I hope this helps. Please let me know if you have any further questions.

Read More

Answer

Thanks for your enquiry.

If you could send along the lot number Iwill be able to provide the antibody concentration.

Read More

Answer

Thank you for your enquiry.

I think the recommendation to use antigen retrieval listed for ICC/IFis a mistake. Antigen retrieval should not be necessary for ICC. Here is a link to a good general ICC protocol on our website if you need it:

https://www.abcam.com/index.html?pageconfig=resource&rid=11417

I hope this is helpful. Please contact me again if you have any further questions.

Read More

Question
Answer

Thank you for your enquiry.

The rabbit monoclonal isotype control antibody that was used in this flow experiment is not available on our catalog. We do have a rabbit polyclonal isotype control, https://www.abcam.com/Rabbit-polyclonal-IgG-ab27478.html, which would still be a reasonable control.

I hope this is helpful. Please contact us again if you have any further questions.

Read More

Answer

Thank you for your enquiry.

On our online datasheet for ab45178, there is a FACS figure with the accompanying figure legend:

"Overlay histogram showing HeLa cells stained with ab45178 (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 (ab45178, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed."

Also, there is 1 Abreview regarding Flow cyt that may also contain some useful protocol information.

Both details above can be found on the online product datasheet at: https://www.abcam.com/ASK1-antibody-EP553Y-ab45178.html

I hope this is helpful. Please contact me if you have any further questions.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Mouse Cell lysate - whole cell (Bone-marrow derived dendritic cells)
Loading amount
70 µg
Specification
Bone-marrow derived dendritic cells
Gel Running Conditions
Reduced Denaturing (8% SDS gel)
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Dr. Caglar Cekic

Verified customer

Submitted May 13 2009

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Mouse Cell lysate - whole cell (neuron)
Loading amount
20 µg
Specification
neuron
Gel Running Conditions
Reduced Denaturing
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 37°C

Abcam user community

Verified customer

Submitted Jun 03 2008

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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