Key Product Details

Validated by

Biological Validation

Species Reactivity

Validated:

Human

Cited:

Human

Applications

Validated:

Immunohistochemistry, Immunohistochemistry-Paraffin, Western Blot, Immunocytochemistry/ Immunofluorescence, Simple Western, Knockdown Validated

Cited:

Immunohistochemistry-Paraffin, Western Blot

Label

Unconjugated

Antibody Source

Monoclonal Mouse IgG1 kappa Clone # LAP6 96-2-22

Format

BSA Free
View all Caspase-9 Primary Antibodies »

Product Specifications

Immunogen

Recombinant human Caspase 9 pro-domain protein. [UniProt# P55211]

Localization

Cytoplasmic and Mitochondrial

Clonality

Monoclonal

Host

Mouse

Isotype

IgG1 kappa

Scientific Data Images for Caspase-9 Antibody (LAP6 96-2-22) - BSA Free

Western Blot: Caspase-9 Antibody (LAP6 96-2-22)BSA Free [NB500-209]

Western Blot: Caspase-9 Antibody (LAP6 96-2-22)BSA Free [NB500-209]

Western Blot: Caspase 9 Antibody (LAP6 96-2-22) [NB500-209] - Western blot analysis of Jurkat (A) and HeLa (B) cell lysate using Caspase 9 (NB500-209) antibody at 1:500.
Immunocytochemistry/ Immunofluorescence: Caspase-9 Antibody (LAP6 96-2-22) - BSA Free [NB500-209]

Immunocytochemistry/ Immunofluorescence: Caspase-9 Antibody (LAP6 96-2-22) - BSA Free [NB500-209]

Immunocytochemistry/Immunofluorescence: Caspase 9 Antibody (LAP6 96-2-22) [NB500-209] - The Caspase 9 antibody was tested in HeLa cells against Dylight 488 (Green). Alpha-tubulin and nuclei were counterstained against Dylight 550 (Red) and DAPI (Blue).
Immunohistochemistry-Paraffin: Caspase-9 Antibody (LAP6 96-2-22) - BSA Free [NB500-209]

Immunohistochemistry-Paraffin: Caspase-9 Antibody (LAP6 96-2-22) - BSA Free [NB500-209]

Immunohistochemistry-Paraffin: Caspase 9 Antibody (LAP6 96-2-22) [NB500-209] - IHC analysis of paraffin-embedded human breast carcinomausing Caspase 9 antibody at 1:25. Image from verified customer review.
Western Blot: Caspase-9 Antibody (LAP6 96-2-22)BSA Free [NB500-209]

Western Blot: Caspase-9 Antibody (LAP6 96-2-22)BSA Free [NB500-209]

Western Blot: Caspase 9 Antibody (LAP6 96-2-22) [NB500-209] - The pro and active forms of Caspase 9 detected in active 293 cell lysate (lane 2). Lane 1: inactive 293 cell lysate.
Simple Western: Caspase-9 Antibody (LAP6 96-2-22)BSA Free [NB500-209]

Simple Western: Caspase-9 Antibody (LAP6 96-2-22)BSA Free [NB500-209]

Simple Western: Caspase-9 Antibody (LAP6 96-2-22) [NB500-209] - Western lane view shows lysates of Jurkat human acute T cell leukemia cell line untreated (-) or treated (+) with 1 mM Staurosporine (STS) for 3 hours, loaded at 0.2 mg/mL. A specific band was detected for Caspase 9 at approximately 53 kDa (as indicated) using 20 ug/mL of Mouse Anti-Caspase 9 Monoclonal Antibody (Catalog # NB500-209). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

Applications for Caspase-9 Antibody (LAP6 96-2-22) - BSA Free

Application
Recommended Usage

Immunocytochemistry/ Immunofluorescence

2-10 ug/ml

Immunohistochemistry

8-25 ug/ml. Use reported by customer review

Immunohistochemistry-Paraffin

8-25 ug/ml

Simple Western

20 ug/ml

Western Blot

1 ug/ml
Application Notes
In Western Blot, bands at ~46kDa-48kDa and 35kDa are seen representing the pro and active forms of Caspase 9, respectively. In ICC/IF cytoplasmic staining can be seen in HeLa cells.

Reviewed Applications

Read 4 reviews rated 4.8 using NB500-209 in the following applications:

Formulation, Preparation, and Storage

Purification

Protein A or G purified

Formulation

PBS

Format

BSA Free

Preservative

0.02% Sodium Azide

Concentration

1.0 mg/ml

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Aliquot and store at -20C or -80C. Avoid freeze-thaw cycles.

Background: Caspase-9

Among the initiator caspases (caspases -2, -8 and -9), caspase-9 is an essential enzymes required for intrinsic or mitochondrial apoptotic pathway. Caspase 9 is a heterotetramer that consists of two anti-parallel arranged heterodimers, each one formed by a 35 kD (p35) and a 10 kD (p10) subunit, and it interacts with several proteins including APAF1, EFHD2, HAX1, BIRC2/c-IAP1, XIAP/BIRC4, BIRC5/survivin, BIRC6/bruce, BIRC7/livin etc. During apoptosome-dependent apoptotic execution, cytochrome-c is released from the mitochondria into cytosol where it binds/activates Apaf-1. Upon nucleotide exchange in the presence of dATP, Apaf-1 oligomerizes into heptameric apoptosome complex and procaspase-9 is recruited to apoptosome where it gets activated. Active caspase-9 at the apoptosome proteolytically activates procaspase-3 (the critical apoptotic executioner caspases) and cleaves poly(ADP-ribose) polymerase (PARP). Both caspases-9 and -3 can be inhibited by selected members of IAPs, with XIAP being their most potent natural inhibitor. Phosphorylation of a major site, Thr125 by CDK1-cyclin B1 leading to inhibition of caspase 9 plays a critical role in the restraint of apoptosis during mitotic arrest. This site is also targeted by ERK1/2 in response to growth factor signalling and by DYRK1A, which plays important roles during development.

Alternate Names

CASP9, Caspase9

Entrez Gene IDs

842 (Human)

Gene Symbol

CASP9

UniProt

P55211

Additional Caspase-9 Products

Product Documents for Caspase-9 Antibody (LAP6 96-2-22) - BSA Free

Certificate of Analysis

To download a Certificate of Analysis, please enter a lot or batch number in the search box below.

Download SDS

Product Specific Notices for Caspase-9 Antibody (LAP6 96-2-22) - BSA Free

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Related Research Areas

Cysteine Proteases and Regulators

Citations for Caspase-9 Antibody (LAP6 96-2-22) - BSA Free

Customer Reviews for Caspase-9 Antibody (LAP6 96-2-22) - BSA Free (4)

4.8 out of 5
4 Customer Ratings
5 Stars
75%
4 Stars
25%
3 Stars
0%
2 Stars
0%
1 Stars
0%

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Customer Images

Showing  1 - 4 of 4 reviews Showing All
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  • Name: Bryan Tinsley
    Application: Immunohistochemistry-Paraffin
    Sample Tested:
    Species: Human
    Verified Customer | Posted 08/29/2014
    IHC analysis of paraffin-embedded human breast carcinoma
    Caspase-9 Antibody (LAP6 96-2-22) - BSA Free NB500-209
  • Name: Bryan Tinsley
    Application: Western Blot
    Sample Tested:
    Species: Human
    Verified Customer | Posted 08/29/2014
    Western blot analysis of extracts from C6 cells at 1:1000
    Caspase-9 Antibody (LAP6 96-2-22) - BSA Free NB500-209
  • Name: Anonymous
    Application: Western Blot
    Sample Tested: COLO-205 Cell Line
    Species: Human
    Verified Customer | Posted 01/13/2012
    Caspase-9 Antibody (LAP6 96-2-22) - BSA Free NB500-209
  • Name: Anonymous
    Application: Western Blot
    Sample Tested: Human cell
    Species: Human
    Verified Customer | Posted 12/23/2011

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Protocols

View specific protocols for Caspase-9 Antibody (LAP6 96-2-22) - BSA Free (NB500-209):


Immunocytochemistry Protocol

Culture cells to appropriate density in 35 mm culture dishes or 6-well plates.

1. Remove culture medium and add 10% formalin to the dish. Fix at room temperature for 30 minutes.
2. Remove the formalin and add ice cold methanol. Incubate for 5-10 minutes.
3. Remove methanol and add washing solution (i.e. PBS). Be sure to not let the specimen dry out. Wash three times for 10 minutes.
4. To block nonspecific antibody binding incubate in 10% normal goat serum from 1 hour to overnight at room temperature.
5. Add primary antibody at appropriate dilution and incubate at room temperature from 2 hours to overnight at room temperature.
6. Remove primary antibody and replace with washing solution. Wash three times for 10 minutes.
7. Add secondary antibody at appropriate dilution. Incubate for 1 hour at room temperature.
8. Remove antibody and replace with wash solution, then wash for 10 minutes. Add Hoechst 33258 to wash solution at 1:25,0000 and incubate for 10 minutes. Wash a third time for 10 minutes.
9. Cells can be viewed directly after washing. The plates can also be stored in PBS containing Azide covered in Parafilm (TM). Cells can also be cover-slipped using Fluoromount, with appropriate sealing.

*The above information is only intended as a guide. The researcher should determine what protocol best meets their needs. Please follow safe laboratory procedures.


Western Blot Protocol

1. Perform SDS-PAGE on samples to be analyzed, loading 10-25 ug of total protein per lane.
2. Transfer proteins to PVDF membrane according to the instructions provided by the manufacturer of the membrane and transfer apparatus.
3. Stain the membrane with Ponceau S (or similar product) to assess transfer success, and mark molecular weight standards where appropriate.
4. Rinse the blot TBS -0.05% Tween 20 (TBST).
5. Block the membrane in 5% Non-fat milk in TBST (blocking buffer) for at least 1 hour.
6. Wash the membrane in TBST three times for 10 minutes each.
7. Dilute primary antibody in blocking buffer and incubate overnight at 4C with gentle rocking.
8. Wash the membrane in TBST three times for 10 minutes each.
9. Incubate the membrane in diluted HRP conjugated secondary antibody in blocking buffer (as per manufacturer's instructions) for 1 hour at room temperature.
10. Wash the blot in TBST three times for 10 minutes each (this step can be repeated as required to reduce background).
11. Apply the detection reagent of choice in accordance with the manufacturers instructions.

Find general support by application which include: protocols, troubleshooting, illustrated assays, videos and webinars.

FAQs for Caspase-9 Antibody (LAP6 96-2-22) - BSA Free

Showing  1 - 2 of 2 FAQs Showing All

  • Q: Could I use HeLa cell lysate as a positive control for Caspase 9 primary antibodies in WB?

    A: After looking through our testing it does appear you should be fine using HeLa as a positive control cell lysate for Caspase 9 in Western Blot, as we have used this sample successfully in our QC tests and shown expression of Caspase 9.

  • Q: Does NB500-209 both recognize human and rat active form of caspase 9 (subunit p35 or subunit p10)? Besides that please let me know if NB500-209 also recognize pro-form caspase 9?

    A: Caspase 9 Antibody (LAP6 96-2-22) # NB500-209 is human specific and do not recognize rat samples. It is expected to detect pro as well as activated form of caspase 9. In our QC data using 293 cell lysates, we observed active subunit p35 only not subunit p10. Both of these antibodies were tested on lysates of 293 cells of human origin (also called Human Embryonic Kidney 293 cells). We did not validate them on rat samples as the antibodies are Human specific.

  • Q: Could I use HeLa cell lysate as a positive control for Caspase 9 primary antibodies in WB?

    A: After looking through our testing it does appear you should be fine using HeLa as a positive control cell lysate for Caspase 9 in Western Blot, as we have used this sample successfully in our QC tests and shown expression of Caspase 9.

  • Q: Does NB500-209 both recognize human and rat active form of caspase 9 (subunit p35 or subunit p10)? Besides that please let me know if NB500-209 also recognize pro-form caspase 9?

    A: Caspase 9 Antibody (LAP6 96-2-22) # NB500-209 is human specific and do not recognize rat samples. It is expected to detect pro as well as activated form of caspase 9. In our QC data using 293 cell lysates, we observed active subunit p35 only not subunit p10. Both of these antibodies were tested on lysates of 293 cells of human origin (also called Human Embryonic Kidney 293 cells). We did not validate them on rat samples as the antibodies are Human specific.

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