11, (), Application(s): Measurement of intracellular cAMP levels in rat 15, (), Application(s): ELISA determining cAMP concentration in. Introduction. The cyclic AMP Enzyme-Linked Immunosorbent Assay (ELISA) kit is a competitive measurement of intracellular cAMP in tissues and cell cultures. cAMP Assay Kit ab uses a sensitive colorimetric competitive ELISA method to The cAMP assay protocol uses a well plate supplied coated with Protein G. The If you are using fluorometric detection, we recommend cAMP Assay Kit It plays a key role as an intracellular second messenger for transduction.
the cAMP intracellular concentration Determination (ELISA). of
Datasheet References 19 Protocols Overview. It plays a key role as an intracellular second messenger for transduction events that follow a number of extracellular signals. This product has been referenced in: Free Radic Biol Med D1 dopamine receptor is involved in shell formation in larvae of Pacific oyster Crassostrea gigas.
Dev Comp Immunol Submit a review Submit a question. Highest votes Sort by: Lowest votes Sort by: Newest First Sort by: We recommend starting with 10 Million cells or mg of tissue.
The kit was easier to use and worked as expected. Directions for sample prep were followed and we had measurable values of cAMP on this assay. The protocols are fairly simple but required thorough review to understand certain steps. Our sample pool in serial dilution performed similar to the standards, however need to be diluted less than the kit recommends for our sample type. We will continue using this assay in the future. Abcam user community Verified customer Submitted Sep 21 Sam Walker Abcam Scientific Support.
Padamjeet Singh Abcam Scientific Support. The hemoglobin will not bind to the antibody in the plate. Results described above raised a key question regarding the signaling pathways leading to sperm capacitation. Because it is now accepted that cAMP signaling is compartmentalized 46 , one possibility consistent with these observations is that distinct molecules involved in the cAMP pathway localize to different sperm compartments.
PNA was used to follow the acrosomal status. Differential interference contrast DIC microscopy was used to control for sperm morphology.
Images are representative of at least three experimental replicates. B, immunodetection of the catalytic subunit of PKA by Western blotting. Only one band of the expected molecular weight is observed. C, immunolocalization of cPKA in mature sperm. When used for immunofluorescence, these antibodies indicated that PKA localized exclusively to the flagellum compartment Fig. To further investigate this hypothesis, sperm heads and tails were obtained from capacitated sperm as described previously As control, capacitated whole-cell sperm extracts were also used.
Each fraction was evaluated for its ability to respond to FK in two complementary assays. Altogether, these experiments are in agreement with the hypothesis that different cAMP pathways are compartmentalized in sperm.
To evaluate this possibility, measurements of the exocytotic acrosome reaction were conducted using Coomassie Blue staining analyses. When sperm were incubated in conditions that support capacitation, FK induced a concentration-dependent increase in the percentage of sperm undergoing the acrosome reaction Fig.
This effect was blocked in the presence of SQ As expected, this inhibitor did not impair acrosomal exocytosis triggered by calcium ionophore A The number of responsive cells is shown in Fig. A, FK induces acrosome reaction. Mouse sperm were incubated under capacitating conditions for 1 h and then further incubated for 30 min in the absence or presence of the tmAC inhibitor SQ Subsequently, either FK or calcium ionophore A A was added, and sperm were incubated for an additional 30 min.
Arrows indicate agonist application. Since its discovery by Rall and Sutherland in the s 49 , 50 , cAMP has been shown to be essential for the regulation of many cell signaling pathways. In mammalian sperm, cAMP has been reported to be involved in the regulation of several capacitation-associated processes such as motility hyperactivation 5 , hyperpolarization of the sperm plasma membrane 51 , 52 , and the increase in protein tyrosine phosphorylation Because of its relevance for cell processes, cAMP levels are tightly regulated both temporally and spatially by different types of adenylyl cyclases and cAMP phosphodiesterases 53 — Two families of adenylyl cyclases are responsible for cAMP synthesis in animals as follows: Adcy10 and tmACs are regulated by different pathways.
Pharmacological and genetic experiments have conclusively demonstrated the essential role of Adcy10 for sperm function 13 , 14 , In sperm, ADP-ribosylation by either pertussis or cholera toxins was used previously to investigate the presence of these G protein subunits in mammalian sperm.
Although originally Hildebrandt et al. Finally, Spehr et al. These results were performed in parallel using brain membrane extracts as positive controls, and similar results were obtained. Although essential for the tmAC signaling pathway, finding the stimulatory G proteins in sperm does not directly imply that tmACs are present in these cells.
It is not surprising then that FK has been a useful tool to evaluate the presence of tmACs in sperm. Similar to the case of G s , different results have been reported concerning the use of FK. Although several groups failed to detect cAMP elevation when the sperm were exposed to FK 8 , 13 , 22 , 27 , 28 , 68 , others have shown that FK increased cAMP levels in sperm 15 , 17 , Consistent with these last studies, Leclerc and Kopf 20 , 69 reported that FK significantly increased adenylyl cyclase activity in membrane fractions of capacitated mouse sperm.
The same authors observed regulation by GTP analogs as well as with mastoparan, a wasp venom toxin known to activate G proteins. Finally, we have performed RT-PCR for each member of the adenylyl cyclase superfamily in testis of mice from different ages and in sperm from adult mice.
Although Sertoli and Leydig cells are present in all stages, their contribution is known to decrease in an age-dependent manner. At 7 days, only pre-meiotic spermatogonia contribute to mRNA expression; at 14 days, most of the germ cells are meiotic spermatocytes, and at 21 days, only postmeiotic spermatids are detected.
In sperm, we were able to detect Adcy6—9, plus the already known sAC Adcy From these, the Adcy8 expression pattern indicates that this enzyme is expressed postmeiotically and that it is developmentally regulated during spermatogenesis.
Because sperm are transcriptionally and translationally inactive, those mRNA transcripts found in isolated mature sperm correspond to transcripts that are present during spermiogenesis in postmeiotic spermatids. However, we cannot discard that other tmACs that are expressed in different stages of germ cells remain in the sperm after translation has occurred in testicular germ cells. In regard to the reproductive phenotype, only Adcy10 -null 13 and Adcy3 -null 17 mice have been reported to be sterile.
Adcy7 knock-out shows partial prenatal and complete postnatal lethality 70 , and null mice for Adcy2 and Adcy9 are not available. Therefore, the reproductive phenotype for these knock-out mice is not known. Knock-out mice for Adcy1 , -5 , -6 , and -8 are fertile; however, it is important to consider that tmACs have overlapping functions in many cells making it difficult to pinpoint the specific isoforms involved in a given process.
Altogether, these data are consistent with the presence of tmACs in mouse sperm. The advantage of this strategy is that the activity of a transmembrane cyclase was not underestimated by Adcy10 activity. Our results also indicate that the PKA catalytic subunit is only present in the sperm flagellum, in agreement with a recent proteomic analysis by Aitken and co-workers Consistent with this differential localization, FK did not activate PKA-dependent phosphorylation in mouse sperm.
After head and tail separation, FK did not increase cAMP levels in the flagellum fractions but was able to induce cAMP accumulation in the head fractions. Altogether, these results suggest that cAMP-dependent pathways are compartmentalized in the sperm. Our working model Fig. Proposed model for the spatial distribution of cAMP in sperm functions. A, Adcy10 mediates activation of flagellum cAMP-dependent pathways such as the activation of PKA is upstream of the increase in tyrosine phosphorylation.
What is the function of tmACs in the sperm? In addition to the well established function in capacitation, cyclic AMP has been proposed to have a role in the sperm acrosome reaction 47 , 48 , 69 , In this study, we provide evidence showing that FK induces the acrosome reaction in capacitated mouse sperm and that the FK effect is blocked in the presence of SQ B The graphical representation of the data arranged accoring to a sigmoidal log-transformed 4-parameter logistic function following log conversion of backfitted cAMP concentrations.
Beavo J, Brunton L. Cyclic nucleotide research -- still expanding after half a century. Nat Rev Mol Cell Biol. Regulation and organization of adenylyl cyclases and cAMP. Houslay M, Adams D. Soderling S, Beavo J.
cAMP Assay Kit (Competitive ELISA) (ab65355)
Cyclic nucleotide ELISA kits tailored to provide highly sensitive results for accurate determination of intracellular and extracellular cAMP/cGMP. Non-linear regression analysis found the ELISA and FRET data to be the correlation of FRET signals to intracellular cAMP concentration over. Does anyone have a detailed protocol for measuring cyclic AMP in mouse I have to measure cAMP in WT mouse islets, and I am using a competitive ELISA assay. procedures or processing steps missing that led to very low measurement. Otherwise, if you are doing intracellular cAMP assay, you can also suspend.