Any reason for cleaved caspase 3 to be seen as purely nuclear staining or is this just an artifact?

Caspase-3 can translocate from the cytoplasm to the nucleus, usually during apoptosis. We do offer a 100% guarantee that this antibody will be useful as stated on the datasheet also.

The datasheet says that it reacts with the active form of Caspase 3 (17 kDa protein), but every time when I do the WB, I get two bands between 15kDa and 20kDa. The different company's cleaved caspase 3 antibody reacts with 17 and 19 kDa protein, so I wonder which band is specific to yours.

Regarding your question, NB600-1235 has been exclusively validated for IHC, not western blot and it is supposed to detect the active form of caspase3. The inactive Caspase 3 is a 31 kD protein, a heterotetramer that consists of two anti-parallel arranged heterodimers, each one formed by a 17 kDa (p17) and a 12 kDa (p12). When it is Cleaved by granzyme B, it generates the two active subunits. As a result the two bands you are observing on the gel should be correct. However, we haven't tested this antibody for western blot.

I'd like to get the information about the active/cleaved Caspase-3 (NB600-1235). The datasheet says that it reacts with the active form of Caspase-3 (17 kDa protein), but every time when I do the WB, I get two bands between 15kDa and 20kDa. The different company's cleaved caspase 3 antibody reacts with 17 and 19 kDa protein, so I wonder which band is specific to yours?

According to the data I could find for the active/cleaved caspase 3, the active form should appear at approximately 17kDa and also has a smaller subunit seen at approximately 12kDa. The inactive form is approximately 32kDa. These are the theoretical weights and can vary based on type of gel and samples they are run on. In most cases, it looks like most antibodies detect the active forms at 19 and 17kDa respectively. With this particular product, NB600-1235, we have not yet validated it for use in western blot, so we unfortunately do not have any information for the location of bands.

This is a Rabbit antibody right so Do you think I can use it on rabbit tissue?

This antibody does react to rabbit but if you are using it with an anti-rabbit secondary, you will almost certainly see some background staining. I know that mouse-on-mouse blocking systems exist, but I am not sure if there is a rabbit equivalent at this time. The best way to use NB600-1235 on rabbit tissues would be to have it directly conjugated to biotin, HRP, or whatever detection method you are planning to use. This will eliminate the need for an anti-rabbit secondary and should give you little to no background. We do offer a custom conjugation service, so if you would like to have this antibody conjugated, please send an email to let me know what you would like to have this antibody conjugated to and I can get you a quote.

Caspase-3 Antibody

Name: Caspase-3 Antibody
Brand: Novus Biologicals
SKU: NB600-1235
Rating: 4 (1 reviews)

Novus Biologicals, part of Bio-Techne | Catalog # NB600-1235

(1)

Citations (39)

Product Datasheet / COA / SDS

100% Guarantee

Product Specifications
Scientific Data
Applications
Preparation & Storage
Background
Product Documents
Specific Notices

Key Product Details

Species Reactivity

Validated:

Human, Fish

Cited:

Human, Mouse, Rat, Bovine, Canine, Fish, Insect - Drosophila melanogaster, Primate, Rabbit

Applications

Validated:

Immunocytochemistry/ Immunofluorescence, Immunohistochemistry, Immunohistochemistry Free-Floating, Immunohistochemistry-Frozen, Immunohistochemistry-Paraffin, Western Blot

Cited:

IF/IHC, Immunocytochemistry/ Immunofluorescence, Immunohistochemistry, Immunohistochemistry Free-Floating, Immunohistochemistry-Paraffin, Western Blot

Label

Unconjugated

Antibody Source

Polyclonal Rabbit IgG

Concentration

1.0 mg/ml

View all Caspase-3 Antibodies »

Product Specifications

Immunogen

This Caspase-3 Antibody was developed against a synthetic peptide corresponding to the cleavage site of human caspase 3 (amino acids 167-175), conjugated to KLH.

Reactivity Notes

Fish reactivity reported in scientific literature (PMID: 28675853).

Localization

Cytoplasmic and some nuclear.

Specificity

This reacts with the active form of Caspase 3 (17 kDa protein).

Clonality

Polyclonal

Host

Rabbit

Isotype

IgG

Theoretical MW

31.7 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Scientific Data Images for Caspase-3 Antibody

Immunohistochemistry-Paraffin: Caspase-3 Antibody [NB600-1235]

Immunohistochemistry-Paraffin: Caspase-3 Antibody [NB600-1235] - Formalin fixed paraffin embedded human tonsil stained with Caspase-3 Antibody (NB600-1235).

Immunohistochemistry-Paraffin: Caspase-3 Antibody [NB600-1235]

Immunohistochemistry-Paraffin: Caspase-3 Antibody [NB600-1235] - Human tonsil.

Immunohistochemistry: Caspase-3 Antibody [NB600-1235] -

Immunohistochemistry: Caspase-3 Antibody [NB600-1235] - Stained sections of fish intestine and liver of the control group showed negative staining reactions in all the examined parts of the intestinal and hepatic tissue (black and red arrows) against caspase-3 (NB600-1235) and TNF-alpha (NBP2-61611) antibodies. Nearly 10% of hepatic portal cells reacted positively to caspase-3. Scale bars = 10 um. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/35624801/) licensed under a CC-BY license.

Applications for Caspase-3 Antibody

Application

Recommended Usage

Immunocytochemistry/ Immunofluorescence

1:10-1:500

Immunohistochemistry

1:10-1:500

Immunohistochemistry-Paraffin

1:50-1:100

Application Notes

IHC-P: recommended pretreatment of citrate buffer, pH 6.0. Recommended incubation time of 30 min at RT. Use in ICC/IF was reported in the scientific literature (PMID: 23813946). Use in Western blot reported in scientific literature (PMID: 29977195). Use in IHC-FrFl reported in secitific publication PMID: 32651317. This Caspase-3 Antibody is validated for IHC-Fr from a verified customer review.

Reviewed Applications

Read 1 review rated 4 using NB600-1235 in the following applications:

Immunohistochemistry-Frozen (1 Review)

Formulation, Preparation, and Storage

Purification

Affinity purified

Formulation

PBS (pH 7.4), 0.2% BSA, Tween-20

Preservative

0.05% 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

Store at 4C. Do not freeze.

Background: Caspase-3

CPP32 encodes a member of the cysteine-aspartic acid protease (caspase) family that plays a key role in apoptosis. Caspase 3 (also known as CPP32, Apopain, Yama, and PARP cleavage protease) is the most extensively studied apoptotic protein among caspase family members (1-2). Caspase 3 is synthesized as an inactive proenzyme that is processed in cells undergoing apoptosis by self-proteolysis and/or cleavage by other upstream proteases (e.g. Caspases 8, 9 and 10). Alternative splicing of CPP32 results in two transcript variants which encode the same protein. The processed form of Caspase 3 consists of large (theoretical molecular weight 17kD) and small (theoretical molecular weight 12kD) subunits which associate to form an active enzyme. Caspase 3 is cleaved at Asp28/Ser29 and Asp175/Ser176. The active Caspase 3 proteolytically cleaves and activates other caspases (e.g. Caspases 6, 7 and 9), as well as relevant targets in the cells (e.g. PARP and DFF). Variations in levels of Caspase 3 have been reported in cells of short-lived nature and those with a longer life cycle. Caspase 3 is the predominant caspase involved in the cleavage of amyloid beta 4A precursor protein, which is associated with neuronal death in Alzheimer's disease (3).

References

1.Mu, N., Lei, Y., Wang, Y., Wang, Y., Duan, Q., Ma, G., . . . Su, L. (2019). Inhibition of SIRT1/2 upregulates HSPA5 acetylation and induces pro-survival autophagy via ATF4-DDIT4-mTORC1 axis in human lung cancer cells. Apoptosis, 24(9-10), 798-811. doi:10.1007/s10495-019-01559-3

2.Sun, C. M., Enkhjargal, B., Reis, C., Zhou, K. R., Xie, Z. Y., Wu, L. Y., . . . Zhang, J. H. (2019). Osteopontin attenuates early brain injury through regulating autophagy-apoptosis interaction after subarachnoid hemorrhage in rats. CNS Neurosci Ther, 25(10), 1162-1172. doi:10.1111/cns.13199

3.Louneva, N., Cohen, J. W., Han, L. Y., Talbot, K., Wilson, R. S., Bennett, D. A., . . . Arnold, S. E. (2008). Caspase-3 is enriched in postsynaptic densities and increased in Alzheimer's disease. Am J Pathol, 173(5), 1488-1495. doi:10.2353/ajpath.2008.080434

Alternate Names

Apopain, CASP3, Caspase3, CPP32, LICE-1, YAMA

Entrez Gene IDs

836 (Human); 12367 (Mouse); 25402 (Rat)

Gene Symbol

CASP3

UniProt

P42574 (Rabbit)

Additional Caspase-3 Products

Product Documents for Caspase-3 Antibody

Product Datasheets

Download Product Datasheets

COA

SDS

Download SDS

Product Specific Notices for Caspase-3 Antibody

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.

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Caspase-3 Antibody

Novus Biologicals, part of Bio-Techne | Catalog # NB600-1235

Key Product Details

Species Reactivity

Validated:

Cited:

Applications

Validated:

Cited:

Label

Antibody Source

Concentration

View all Caspase-3 Antibodies »

Product Specifications

Immunogen

Reactivity Notes

Localization

Specificity

Clonality

Host

Isotype

Theoretical MW

Scientific Data Images for Caspase-3 Antibody

Immunohistochemistry-Paraffin: Caspase-3 Antibody [NB600-1235]

Immunohistochemistry-Paraffin: Caspase-3 Antibody [NB600-1235]

Immunohistochemistry: Caspase-3 Antibody [NB600-1235] -

Applications for Caspase-3 Antibody

Immunocytochemistry/ Immunofluorescence

Immunohistochemistry

Immunohistochemistry-Paraffin

Reviewed Applications

Formulation, Preparation, and Storage

Purification

Formulation

Preservative

Concentration

Shipping

Stability & Storage

Background: Caspase-3

Alternate Names

Entrez Gene IDs

Gene Symbol

UniProt

Additional Caspase-3 Products

Product Documents for Caspase-3 Antibody

Product Datasheets

COA

Certificate of Analysis

SDS

Product Specific Notices for Caspase-3 Antibody