The use of apoptosis antibodies and controls in cell death research

Mon, 03/20/2017 - 15:06

Apoptosis is a method of programmed cell death that is notably characterized by a morphological change in cellular nuclei and membrane appearance.  Not to be confused with necrosis, apoptosis is a pathway that is induced by a variety of factors that activate cysteine proteases known as caspases to lead the cell to its ultimate death versus natural death of a cell. While excessive apoptosis can be detrimental, it can also be part of our immune response or in protection of cells that have been damaged by disease or toxic stimuli.  Using antibodies against apoptotic pathway proteins is an effective way to investigate the role of apoptosis in a number of experimental models.  However, it is important to use antibodies against well-defined key apoptotic players and to integrate controls when possible. 

Caspase 3 antibody

Caspase‑3 was detected in immersion fixed Jurkat human acute T cell leukemia cell line stimulated with staurosporin using Human/Mouse Active Caspase‑3 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF835) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Rabbit IgG Secondary Antibody (yellow; Catalog # NL004) and counterstained with DAPI (blue). View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

Novus Biologicals carries a variety of verified apoptosis marker antibodies, including Caspase 3/Cleaved Caspase 3, Caspase 7, Caspase 9 and PARP.  Caspase 3 is a highly important enzyme responsible for cleaving essential apoptotic proteins, including PARP.  In fact, Fassl et al used the Caspase 3 antibody in western blot as an apoptotic indicator control in glioblastoma lysates.  The cleaved form of Caspase 3 may also be of interest depending on which stage of apoptosis is being investigated, and Janek et al used a Cleaved Caspase 3 antibody in immunofluorescence to show that treatment of melanoma A375 cells with PFII induced a condensed round and blebbing membrane formation with disorganized F-actin.   Next, Caspase 7 is an effective apoptosis antibody to use as a hallmark of apoptosis as well.  Nearly synonymous with Caspase 3, Caspase 7 is specific to the cleavage of some proteins that Caspase 3 is not.  Lubet et al used a Caspase 7 antibody  in immunohistochemistry to study the toxicity of NPX in urinary bladder cancer. 

Next up, Caspase 9 is an apoptosis factor that is induced by the release of cytochrome c from the mitochondria.  Therefore, Caspase 9 may be the best apoptotic indicator to use an antibody for in research examining mitochondrial oxidative stress or related diseases and therapeutics.  Caspase 9 is upstream of caspase 3 and caspase 6 and is vital to normal development during the development of the central nervous system.  Pradhan et al used a Caspase 9 antibody to test caspase-dependent apoptosis in tumor cells exposed to an iron based anti-tumor drug.  Furthermore, PARP is a protein involved in DNA repair.  During apoptosis, PARP is cleaved by caspase 3, which makes a cleaved PARP antibody an efficient way to detect active apoptosis in a cell.  Chen et al used a cleaved PARP antibody in their research of whether emodin induces apoptosis in HL-60 cells.  They found that by using a caspase 3 inhibitor in cell treatments, they saw a reduction in cleaved PARP and overall apoptosis.

In addition to apoptotic antibodies, it is important to use a nuclear and cytoskeletal identifier in experiments to have a visual inspection of cell and nuclear integrity.  The use of DAPI to fluoresce the nucleus is very popular in immunofluorescence, as well as the use of an actin antibody to examine the integrity of the cytoskeleton. 

Learn more about apoptosis with our handy apoptosis handbook.

  1. Porter AG1, Jänicke RU. Emerging roles of caspase-3 in apoptosis. [PMID: 10200555]
  2. Fassl A, Tagscherer KE, Richter J, De-Castro Arce J, Savini C, Rösl F, Roth W. Inhibition of Notch1 signaling overcomes resistance to the death ligand Trail by specificity protein 1-dependent upregulation of death receptor 5. [PMID: 26469969]
  3. Janek T, Krasowska A, Radwańska A, Łukaszewicz M. Lipopeptide biosurfactant pseudofactin II induced apoptosis of melanoma A 375 cells by specific interaction with the plasma membrane. [PMID: 23483962]
  4. Lubet RA, Scheiman JM, Bode A, White J, Minasian L, Juliana MM, Boring DL, Steele VE, Grubbs CJ. Prevention of chemically induced urinary bladder cancers by naproxen: protocols to reduce gastric toxicity in humans do not alter preventive efficacy. [PMID: 25762530]
  5. Pradhan N, Pratheek BM, Garai A, Kumar A, Meena VS, Ghosh S, Singh S, Kumari S, Chandrashekar TK, Goswami C, Chattopadhyay S, Kar S, Maiti PK. Induction of apoptosis by Fe(salen)Cl through caspase-dependent pathway specifically in tumor cells. [PMID: 24804954]
  6. Chen YC, Shen SC, Lee WR, Hsu FL, Lin HY, Ko CH, Tseng SW. Emodin induces apoptosis in human promyeloleukemic HL-60 cells accompanied by activation of caspase 3 cascade but independent of reactive oxygen species production. [PMID: 12445860]

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