BioPath Online

Pathway Focus: Apoptosis

Read Article 1
Role of cyclins and cyclin-dependent kinases (CDK) in the cell cycle - Antibodies for Apoptotis markers | Read More
Read Article 2Multiplexed detection and quantification of apoptosis markers - Luminex® Apoptosis 3-Plex | Read More
Read Article 3TUNEL vision: Image DNA fragmentation - Click-iT® TUNEL assays | Read More
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Role of cyclins and cyclin-dependent kinases (CDK) in the cell cycle -

Two key regulatory molecule classes, cyclins and cyclin-dependent kinases (CDK), determine cell cycle progression. Cyclins form the regulatory subunits and CDK the catalytic subunits of the activated heterodimer. When activated by cyclins, CDKs phosphorylate different targets so cells can enter the cell cycle’s next phase. Different cyclin-CDK combinations determine downstream protein targets, and thus the different phases, in the cell cycle.

Upon receiving the extracellular signal, G1 cyclin-CDK complexes initiate a downstream cascade of events, preparing the cell to go into S phase and start replicating DNA. Other cyclin-CDK complexes trigger other cell cycle stages. Any abnormality in this activation can trigger apoptosis.

Invitrogen offers over 300 antibodies to study the cell cycle, apoptosis, and related pathways. These antibodies include phosphorylation site–specific antibodies, total protein detection antibodies, and cleavage-specific antibodies. These antibodies are validated with multiple applications, and their specificity is determined against multiple species.  Invitrogen offers whole panel of antibodies against cyclins and CDK markers to help researchers learn their roles in cell cycle and apoptosis.




Figure 1.
Immunohistochemical staining of hepatocellular carcinoma tissue using Ms anti-cyclin E (clone HE12).


Figure 2.
Cdk antibody specificity. Extracts of HeLa cells were resolved by SDS-PAGE and transferred to PVDF. The membrane was blocked, then incubated with the Cdk1 [pTpY14/15] antibody following prior incubation with: no peptide (1), the nonphosphopeptide corresponding to the phosphopeptide immunogen (2), a generic phosphotyrosine-containing peptide (3), a generic phosphothreonine-containing peptide (4), or the phosphopeptide immunogen (5). The membrane was then incubated with goat F(ab’)2 anti-rabbit IgG HRP-conjugate and signals were detected using the Pierce SuperSignal™ method.
 
Only the phosphopeptide corresponding to Cdk1 [pTpY14/15] blocks the antibody signal.



ProductTarget proteinClonality, clone (isotype)Reactive speciesApplications
Qty.
 Cat. No.
 
Cyclin D1
Cyclin D1
pAb (Rb IgG)
Hu, Ms, Rt
KA, WB, IHC, IF, IP (co)
500 μg
AHF0102
CDK1 [pTpY14/15]  pAb
Cdk1 [pT14/pY15]
pAb (Rb IgG)
Hu, Ms, Rt
WB, IP
10 blot44686G
Mouse anti-cyclin E
Cyclin E
mAb, HE12 (Ms IgG1-κ)
Hu
WB, IP, IHC, FC
200 μg321600
Cyclin B1 (V152), Ms X
Cyclin B1
mAb, V152 (Ms IgG1)
Hu, Ms, Rt, Ha
WB, IHC, FC, IP (co)
100 μgAHF0052
Cyclin D2 (DCS 3.1), Ms X
Cyclin D2
mAb, DCS-3.1 (Ms IgG2a)
Hu, Ms, Rt
WB, IF, FC, ICC, IP, BB
100 μgAHF0112
Mouse anti-cyclin E
Cyclin E
mAb, HE172 (Ms IgG2b-κ)
Hu
WB, IP, EMSA
200 μg
321500
Rb anti-cyclin D1b
Cyclin D1b 
pAb, ZMD.528 (Rb IgG)
Hu
WB
100 μg
420800
Mouse anti-cyclin D1
Cyclin D1
mAb, DI-72-13G (Ms IgG1)
Ms, Rt
WB, IP, IF, IHC
100 μg
134500
Mouse anti-cyclin A
Cyclin A
mAb, E23 (Ms IgG2a-κ)
Hu, Ms, Rt, Mk, B
E, IP, WB, IF, IHC
100 μg
334900
Cyclin D3 (DCS 22), Ms X
Cyclin D3
mAb, DCS-22 (Ms IgG1)
Hu, Ms, Rt, Mk
WB, IHC, IF, FC, IP
100 μg
AHF0132
Cyclin E (poly)
Cyclin E
pAb (Rb IgG)
Hu
WB, IF, KA, IP (co)
500 μg
AHF0162
CDK1/P34CDC2 Kinase (A17.1.1) Ms X
Cdk1/p34Cdc2
mAb, A17.1.1 (Ms IgG2a)
Hu, Ms, Rt, Gp, Wk, X, Ch
WB, IHC, IF, IP, KA, BB
100 μg
AHZ0122
CDK4 (AB 5) Ms
Cdk4
pAb (Rb IgG)
Ms, Rt
WB, KA, IP (co)
500 μg
AMZ0182

Multiplexed detection and quantification of apoptosis markers

Cytochrome c is a protein that normally resides within mitochondria and is released to the cytosol in response to apoptotic stimuli.
Cleaved caspase-3 [175/176] is an important reporter of initiator caspase activation. Cleaved poly (ADP-ribose) polymerase (PARP) [214/215] is an important reporter of caspase-3 activation.

These three markers are important players in the apoptosis pathway. In apoptosis, cell populations are deleted in response to self-recognition, failure to bind MHC, and cytokine/growth factor withdrawal. This pathway regulates and maintains the immune system.
To facilitate apoptosis detection, we developed a multiplexed sandwich immunoassay for the Luminex® platform that permits the simultaneous detection of cytochrome C, PARP (cleaved), and caspase-3 (cleaved). ELISA kits for each marker are also available for single measurements.  To demonstrate the utility of this system, the three analytes were profiled in Jurkat cell fractions either treated with 1 μM staurosporine to induce apoptosis, or untreated controls. Significant changes in the three analyte profiles were detected within two hours. Linearity of dilution experiments performed to validate this assay system yielded R2 values of 0.99 and higher.


Detection of apoptosis by analysis of various cell fractions.
Caspase-3, cytochrome c, and PARP were measured in whole Jurkat cell lysates, cytosolic fractions, and mitochondrial fractions, and analyzed using the apoptosis 3-Plex.  Two-hour treatment with staurosporine resulted in cleavage of caspase-3 and of PARP.  Increase in cytosolic cytochrome c was accompanied by a decrease in the mitochondrial compartment, consistent with the translocation of this analyte from the mitochondria.



ProductSpeciesQty.
 Cat. No.
 
Apoptosis 3-plex Assay
Hu
100 tests
LHO0007
Caspase-3 (active) ELISA Kit
Hu, Ms, Rt
96 tests
KHO1091
PARP, Cleaved [214/215] ELISA Kit
Hu
96 tests
KHO0741
Cytochrome c ELISA Kit
Hu
96 tests
KHO1051

TUNEL vision: Image DNA fragmentation - Click-iT® TUNEL assays

At the final stage of apoptosis, adherent cells often detach from the slide or well before they can be detected. As a result, accurate detection of DNA fragmentation that is a hallmark as well as the ultimate determinant of apoptosis is often difficult to reproduce. The fast and reliable Click-iT® TUNEL Imaging Assays detect this late-stage apoptosis.

Click-iT® TUNEL Imaging Assays use a dUTP modified with an alkyne, a small, bio-orthogonal functional group that also allows the nucleotide to be more readily incorporated by TdT than other modified nucleotides. The enzymatically incorporated nucleotide is detected with a click reaction—a copper-catalyzed triazole formation between an azide and an alkyne.
A key benefit of click reactions is that the reaction components are inert; no side reactions occur, and the label and detection tags react selectively with one another. Click chemistry-labeled molecules can be applied to complex biological samples and detected with unprecedented sensitivity, thanks to extremely low background. When compared with assays using other modified nucleotides, the Click-iT® TUNEL assay can detect a higher percentage of apoptotic cells under identical conditions within two hours.

Learn more about Click-iT® TUNEL Imaging Assays

Figure 1. Multicolor imaging is a snap with Click-iT® EdU. Muntjac cells were treated with 10 μM EdU for 45 minutes. Cells were then fixed and permeabilized, and EdU that had been incorporated into newly synthesized DNA was detected using the far red–fluorescent Click-iT® EdU Alexa Fluor® 647 Imaging Kit. Tubulin was labeled with a mouse anti-tubulin antibody and visualized with an Alexa Fluor® 350 goat anti–mouse IgG antibody. The Golgi complex was stained with the green-fluorescent Alexa Fluor® 488 conjugate of lectin HPA from Helix pomatia (edible snail), and peroxisomes were labeled with a rabbit anti-peroxisome antibody and visualized with an orange-fluorescent Alexa Fluor® 555 donkey anti–rabbit IgG antibody.


Product Cat. No.
 
Click-iT® EdU Alexa Fluor® 647 Imaging KitC10085
Alexa Fluor® 350 goat anti–mouse IgG antibodyA11045
Alexa Fluor® 488 conjugate of lectin HPA from Helix pomatia (edible snail)L11271
Alexa Fluor® 555 donkey anti–rabbit IgG antibodyA31572