Cell signaling assay kits

 

Cell signaling is a complex communication mechanism that requires multiple players both outside and inside the cell.

Abcam offers a complete range of kits to detect and quantify signaling molecules such as second messengers, MAP kinases and transcriptional factors activators in a variety of essential pathways.

download our How GPCRs work pathway card

A lot of factors are involved in intracellular signaling cascades, but the following players can be found in most signaling pathways:

  1. Signal receptors
  2. Second messengers
  3. Transcription factors
  4. Other factors such as small GTPases and kinases

 

1. Signal receptors

 

Receptors are the first point of contact of the signaling molecule with the target cell. The majority of receptors sit in the cellular membrane, and the largest family of membrane receptors is the G protein coupled receptors (GPCR or seven-transmembrane domain receptors) family. They are coupled to intracellular signaling pathways via a G protein, which is comprised of three subunits - α, β and γ, and possess GTPase activity.

Upon receptor binding the G protein is activated, leading in turn to the generation of intracellular second messengers.

2. Second messengers

 

Second messengers are responsible for relaying the signals received by the membrane receptors to the intracellular downstream factors, as well as amplifying the strength of the signal.

The major classes of second messengers are:

Cyclic nucleotides

 

Cyclic adenosine monophosphate (cAMP) transfers the signal from hormones (adrenaline and glucagon among others) through kinase activation, leading to phosphorylation of target proteins and activation of transcription factors that will change the gene transcription pattern in the cell. cAMP is also implicated in other cellular activities such as inhibition of apoptosis.

Cyclic guanosine monophosphate (cGMP) transfers the signal from ANP (atrial natriuretic peptide) among others through kinase and phosphodiesterases activation. cGMP is implicated in a variety of cellular pathways such as smooth muscle relaxation, cell growth and cell differentiation.

Inositol triphosphate (IP3) and diacylglycerol (DAG)

 

They are signaling lipids that transfer the signal from hormones as well as from neurotransmitters through GPCRs, mainly through PLC (phospholipase C) activation. PLC specifically hydrolyzes PIP2 (phosphatidylinositol biphosphate) producing DAG and IP3. DAG remains in the cell membrane and recruits PKC (protein kinase C) which in turn phosphorylates its target proteins. IP3 diffuses through the cytosol and causes the release of calcium ions from the endoplasmic reticulum into the cytosol, also triggering PKC activation.

Calcium ions (Ca2+)

 

Calcium ions are probably the most widely used intracellular messengers, as they not only respond to PKC activation (as described above) but also to neurotransmitters and muscle contraction, as well as being involved in apoptosis. In response to signal, the levels of intracellular calcium will dramatically increase, leading to activation of the regulatory protein calmodulin. Calmodulin will in turn activate calcium/calmodulin-dependent proteins or directly activate other effector proteins.

Nitric Oxide (NO)

 

Nitric oxide (NO) is a key signaling molecule involved in several physiological processes in the nervous, immune, and cardiovascular systems. NO plays an important role in smooth muscle relaxation and vasodilation through cGMP production. NO is also a neuronal transmitter and it is involved in macrophage cytotoxicity.

 Assay kits available for detection and quantification of second messengers:

Product NameProduct DescriptionProduct code
cAMP Direct Immunoassay Kit Colorimetric quantification of cAMP (0.02μM - 2μM)

ab65355

cAMP Direct Immunoassay Kit (Fluorometric) Fluorometric quantification without acetylation step

ab138880

cGMP Direct Immunoassay Kit Colorimetric quantification of cGMP (8nM - 2μM) ab65356
Calcium Assay (Luminometric) Ca2+ quantification in aequoporin transfected cells

ab112114

Calcium Detection Kit (Colorimetric) Colorimetric quantification of 0.1mM - 25mM Ca2+ ab102505
Calcium Quantification Kit (Fluorometric) Fluorometric quantification of 30μM - 10mM Ca2+ in solution samples ab112115
Fluo-8 Medium Removal Calcium Assay Kit Fluorometric quantification of Ca2+ in cells using Fluo-8 (green dye) ab112128
Fluo-8 No Wash Calcium Assay Kit

Fluorometric quantification of Ca2+ in cells without washing step using Flu-8 (green dye)

ab112129
Rhod-4 Medium Removal No Wash Calcium Assay Kit Fluorometric quantification of Ca2+ in cells using Rhod-4 (red dye) ab112156
Rhod-4 No Wash Calcium Assay Kit Fluorometric quantification of Ca2+ in cells without washing step using Rhod-4 (red dye) ab112157
Nitric Oxide Assay Kit (Fluorometric) Fluorometric quantification of total nitrate/nitrate (NO conversion) ab65327
Nitric Oxide Assay Kit (Colorimetric) Colorimetric quantification of total nitrate/nitrite (NO conversion) ab65328

 

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3. Transcription factors

 

Once the signal has been amplified and transduced by the second messengers and/or signaling kinases, downstream effector proteins will be activated. Many of these downstream effector proteins are transcription factors that will induce transcriptional changes (activation or repression of specific genes) in response to the extracellular signal. They bind to specific sequences called transcription response elements within the promoter region of the target genes, leading to conformational chromatin changes and subsequent activation or silencing of said genes.

Assay kits available for detection of transcription factors:

Product NameProduct DescriptionProduct code
ATF2 (pT69/pT71) Transcription Factor Assay Kit Colorimetric detection of specific ATF2 binding activity to cAMP response elements

ab133074

CREB (pS133) Transcription Factor Assay Kit Colorimetric detection of specific CREB binding activity to cAMP response elements

ab133117

ChREBP Transcription Factor Assay Kit Colorimetric detection of specif ChREBP binding activity to carbohydrate response elements

ab133103

HIF1 alpha Transcription Factor Assay Kit Colorimetric detection of specific HIF1 alpha binding activity to HIF1 alpha response elements ab133104
NFkB p50 Transcription Factor Assay Kit Colorimetric detection of specific NFkB p50 subunit binding activity to NFkB response elements ab133105
NFkB p50/p65 Transcription Factor Assay Kit Colorimetric detection of NFkB binding activity to NFkB response elements ab133128
NFkB p65 Transcription Factor Assay Kit Colorimetric detection of specific NFkB p65 subunit binding activity to NFkB response elements ab133112
PPAR (α, δ, γ) Transcription Factor Assay Kit Colorimetric detection of PPARs binding activity to PPAR response elements ab133113
PPAR alpha (α) Transcription Factor Assay Kit Colorimetric detection of specific PPAR alpha binding activity to PPAR response elements ab133107
PPAR delta (δ) Transcription Factor Assay Kit Colorimetric detection of specific PPAR delta binding activity to PPAR response elements ab133106
PPAR gamma (γ)Transcription Factor Assay Kit Colorimetric detection of specific PPAR gamma binding activity to PPAR response elements ab133101
SREBP1 Transcription Factor Assay Kit Colorimetric detection of specific SREBP1 binding activity to SREBP response elements ab133125
SREBP2 Transcription Factor Assay Kit Colorimetric detection of specific SREBP2 binding activity to SREBP response elements ab133111

 

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4. Small GTPases signaling

Small GTPases are a family of hydrolases that are able to bind and hydrolyze guanosine triphosphate (GTP) to form guanosine diphosphate (GDP). They are similar to the G proteins linked to receptors (mentioned above), but they work as monomers instead of being heterotrimers.

Ras GTPases are the most well-known members of the family and they are key regulators of diverse cellular and developmental events such as differentiation, cell division, vesicle transport, nuclear assembly and control of the cytosketelon. They act as transducer proteins, passing the signal to downstream regulatory factors and effector proteins such as kinases.

Assay kits available for detection of small GTPases: 

Product NameProduct DescriptionProduct code
Rac Activation Kit Semi-quantitative analysis of cellular activation state of Rac1 and Rac2

ab139586

Ral Activation Kit Semi-quantitative analysis of cellular activation state of Ral

ab128506

Rap Activation Kit Semi-quantitative analysis of cellular activation state of Rap

ab128505

Ras Activation Kit Semi-quantitative analysis of cellular activation state of K-Ras, N-Ras and H-Ras ab128504

 

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5. Kinase signaling

Intracellular protein kinases can act as downstream effector for the signal transduced by second messengers and small GTPases.

Second messengers typically activate serine/threonine kinases, which will in turn phosphorylate and activate/inhibit a variety of targets including enzymes, receptors, ion channels and structural proteins. Some of the kinases that act as effectors for some of the messengers previously discussed are PKA (mainly activated through cAMP), PKC (activated through DAG and calcium ions), and CaMKII (activated through calcium/calmodulin).

One of the downstream pathways activated by the Ras GTPase family is the MAP Kinase cascade. MAP Kinases (Mitogen-activated protein kinases or MAPKs) are serine/threonine specific kinases involved in a variety of cellular pathways including proliferation, differentiation, mitosis, gene expression and apoptosis among others. In mammalian cells, there are three major groups of MAP kinases: extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK) and p38 MAP kinase. ERK mediates response to growth factors and is involved in cell proliferation and cell cycle progression. On the other hand, the JNK and p38 pathways mediate response to stress stimuli (cytokines and UV irradiation, for example) and are involved in cell differentiation and apoptosis.

Another important cascade pathway is the PI 3-Kinase signaling cascade which can also be activated by Ras. PI 3-kinases (phosphatidylinositide 3-kinases or PI3Ks) are intracellular signal transducers that generate specific phosphatidylinositols that serve as docking sites in the plasma membrane for signaling proteins such as Akt/PKB.

Assay kits available for detection of signaling kinases:

Product NameProduct DescriptionProduct code
ERK/MAPK (pT202/pY204) Translocation Assay Kit (Cell-based) Qualitative analysis of translocation to/from the nucleus

ab133123

p38 MAPK (pT180/pY182) Translocation Assay Kit (Cell-based) Qualitative analysis of translocation to/from the nucleus

ab133121

JNK Activity Screening Kit WB analysis of JNK activation through pull-down and c-Jun phosphorylation analysis

ab65784

PI 3 Kinase Assay Kit Semi-quantitative analysis of PI3 Kinase activation

ab128503

AKT Activity Assay Kit WB analysis of Akt activation through pull-down and GSK3α phosphorylation analysis ab65786
Universal Kinase Assay Kit (Fluorometric) Fluorometric quantification of activity of any kinase that uses ATP as donor ab138879

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