Overview

  • Product name
  • Description
    Rabbit polyclonal to Epac1
  • Host species
    Rabbit
  • Specificity
    ab21236 does not cross react with Epac2. It has not been tested against endogenous Epac1 but recombinant Epac1.
  • Tested applications
    Suitable for: WB, ELISA, IP, IHC-Fr, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Rabbit, Human
  • Immunogen

    Synthetic peptide:

    DHSVLTLQLPVTASVR

    , corresponding to amino acids 526-541 of Epac1.

  • Positive control
    • Epac1 recombinant protein
  • General notes
    Ab21236 is supplied in an antibody stabilisation buffer. IgG concentration 0.78-0.94mg/ml in antibody stabilisation buffer.

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Upon delivery aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
  • Concentration information loading...
  • Purity
    Immunogen affinity purified
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab21236 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB 1/500. Predicted molecular weight: 99 kDa.
ELISA 1/10000.
IP Use at an assay dependent concentration.
IHC-Fr Use at an assay dependent concentration. PubMed: 18577515
ICC/IF Use a concentration of 5 µg/ml.

Target

  • Relevance
    The activation of RaP1 by cAMP is independent of PKA and is mediated by recently discovered family of guanine nucleotide exchange factors (GEFs) called cAMP-GEFs or Epacs. The Epac signaling therefore represents a novel mechanism for cAMP signaling with in the cAMP cascade. There are 2 members of the Epac family, Epac1 and Epac 2. Both proteins are multidomain proteins containing an autoinhibitory cAMP-binding domain that inhibits the catalytic region and a DEP domain (dishevelled, Egl-10 and pleckstrin homology domain) targeting the membrane anchors. EPAC2 has an additional cAMP-binding site in its N-terminus that binds cAMP with low affinity. EPAC1 mRNA is broadly expressed, with particularly high levels occurring in the thyroid, ovary, kidney and certain brain regions, whereas expression of EPAC2 mRNA appears to be restricted to the brain and adrenal glands. Epac 1 and Epac 2 also interact with light chain 2 (LC2) or MAP1A that serves as a scaffolding structure to stabilize the signal transduction complex. The Epac 1-selective antibodies were generated against unique antigenic sequences form near N-terminus and between RasGEFN and Ras GEF domains. The antibodies to Epac 1are affinity purified over immobilized antigen based chromatography.
  • Cellular localization
    Endomembrane system
  • Database links
  • Alternative names
    • bcm910 antibody
    • CAMP GEFI antibody
    • cAMP regulated guanine nucleotide exchange factor I antibody
    • CAMPGEFI antibody
    • CGEF 1 antibody
    • CGEF1 antibody
    • EPA1 antibody
    • Epac 1 antibody
    • EPAC antibody
    • EPAC1 antibody
    • Exchange factor directly activated by cAMP 1 antibody
    • Exchange protein directly activated by cAMP 1 antibody
    • MGC21410 antibody
    • RAP guanine nucleotide exchange factor antibody
    • Rap guanine nucleotide exchange factor (GEF) 3 antibody
    • RAP guanine nucleotide exchange factor 3 antibody
    • Rap1 guanine nucleotide exchange factor directly activated by cAMP antibody
    • RAPGEF3 antibody
    see all

Images

  • ab21236 staining Epac1 in mouse pancreatic acini tissue section by Immunohistochemistry (Frozen tissue sections). Tissue underwent fixation in 4% paraformaldehyde in PBS pH 7.4 for 30 minutes, PBS washed, cryoprotected and frozen with isopenatane cooled with liquid nitrogen. The primary antibody was diluted 1/200 and incubated with sample for 2 hours at room temperature. A cy3-conjugated donkey polyclonal to rabbit IgG at 1/200 dilution was used as secondary for 1 hour. Prolong Gold with 4,6-diamino-2-phenylindole was added to mounting medium to counterstain nuclei. 

  • ICC/IF image of ab21236 stained MCF7 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab21236, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

References

This product has been referenced in:
  • Garcia-Morales V  et al. The microRNA-7-mediated reduction in EPAC-1 contributes to vascular endothelial permeability and eNOS uncoupling in murine experimental retinopathy. Acta Diabetol 54:581-591 (2017). Read more (PubMed: 28353063) »
  • Tobin GA  et al. The Role of eNOS Phosphorylation in Causing Drug-induced Vascular Injury. Toxicol Pathol 42:709-724 (2014). Read more (PubMed: 24705881) »
See all 9 Publications for this product

Customer reviews and Q&As

1-7 of 7 Abreviews or Q&A

Question
Answer

Thank you for contacting Abcam earlier today.
The composition of antibody stabilization buffer has the following components: the exact concentrations of these constituents is proprietary in nature and cannot be disclosed.
Tris/Glycine buffer pH 7.4, glycerol, stabilizing proteins, cryogenic stabilizers, and preservatives (0.02% sodium azide)
This preparation cannot be used for coupling antibodies to other substrates,.
Please let me know if there is anything else I can help you with.

Read More

Answer

The rabbit monoclonal will be effective for IF. The application field lists ICC (iimunocytochemistry) which is IF (immunofluorescence) if one is staining cell cultures with a fluorochrome.

I have issued a free of charge replacement with ab109415.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

Read More

Answer

Thank you for your thorough reply. One other question I meant to ask was how much sample you load per lane of the gel. We recommend at least 20ug per lane, especially if no signal is evident.

Assuming you are loading enough sample, I suggest trying a different antibody as a free-of-charge replacement for ab21236. We do have another lot of ab21236 but there have been other reports of batch inconsistency and I think a rabbit monoclonal such as ab109415 will be a better choice for you. Please let me know if you would like to try this.

Click here (or use the following: https://www.abcam.com/index.html?datasheet=109415).

Read More

Answer

Thank you for bringing this to our attention. I am sorry to read that the antibody is giving you unexpected results in these two applications.

There may be a modification I can suggest to improve your result but it may be the case that, as you suspect, the antibody is defective. The other possibility, assuming your protocol is correct, is that the samples do not contain Epac1, or in amounts too low for the the antibody to detect. Do you have any other evidence, your own or from the litereature, that your cells contain Epac1?

For our records, could you please answer a few questions about your protocol?

Can you please tell me the species of the endothelial cells, and their origin? That is, is this a primary culture, or a continuous cell line?

Can you please tell me what you used for a blocking solution for the western blotting, and the antibody diluent?

Are you confident the transfer from gel to blot was successful? Finally, are you confident that the secondary antibody is effective?

If I cannot offer any suggestions to improve the protocol, and you are confident the cells express Epac1 protein, I think it will be best to replace the antibody, either with a vial from a different lot, if available, or a different Epac1 antibody. This replacement will be free-of -charge, assuming your samples are of one of the species listed on the datasheet (Human, Mouse, Rat and Rabbit).

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Rabbit Cell lysate - whole cell (pancreatic acini)
Loading amount
20 µg
Specification
pancreatic acini
Gel Running Conditions
Reduced Denaturing (7.5)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Dr. maria sabbatini

Verified customer

Submitted Sep 09 2008

Question

BATCH NUMBER 210603 ORDER NUMBER 250420 DESCRIPTION OF THE PROBLEM No signal: I have used Ab21236 at 1:000 and 1:500 dilution in 0.1%TTBS in a Western blot of HEK 293 cells expressing epac1 with cAMP sensors. I did not obtain any signals other than magic marker molecular weight proteins indicating 2 nd antibody reactivity. I tested the antibody 8/30/07 and again yesterday to confirm results obtained using an anti-epac1 ab from another source. I checked to see if any reports were sent regarding this antibody. No reports were submitted by other investigators. What has been your experience with your antibody. What positive controls are you using? i.e. kidney, cardiac or brain? We are testing your antibody not only against expressed epac1 but looking at mouse cardiac, brain and parotid tissue fractions. SAMPLE HEK-293 recombinant epac1 with cAMP sensors/mouse whole cell lysates, heart, brain, parotid PRIMARY ANTIBODY Ab21236 Abcam/rabbit/0.1%TTBS/1:500, 1:1000/ 1 and 2 hours for primary;1 h for 2ndary. Standard wash protocols using with 0.1 TTBS DETECTION METHOD Amersham ECL/Pierce ECL POSITIVE AND NEGATIVE CONTROLS USED Positive: HEK 293 cells expressing epac1 with cAMP sensors. ANTIBODY STORAGE CONDITIONS -20 C, aliquoted for Ab21236 and -80 C, aliquoted for 2ndary SAMPLE PREPARATION NuPAGE sample buffer at 1x/multiple, group specific protease inhibitors/65 C for 15 min. AMOUNT OF PROTEIN LOADED Sufficient to give strong signals using 3 other antibodies from different sources but with only 77% (or less) homology to mouse epac1. We wanted to test your antibody because your epitope antibody corresponds to 15/16 aa of mouse sequence and more than one immunoreactive peptide was observed between 150 and 100 Kd . Other tissue: mouse whole cell lysates up to 100 micrograms protein gel load. ELECTROPHORESIS/GEL CONDITIONS Tris-Glycine SDS/6%PAGE; reducing TRANSFER AND BLOCKING CONDITIONS 1/2 Towbain/30 volt constant/overnight at 4 C/1%BSA,1%PEG,1%PVP in 0.1%TTBS, 1 h at rt SECONDARY ANTIBODY Jackson ImmunoResearc/Donkey/1:1000, 1:20000/1 hour/ standard wash protocols HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? primary ab dilution

Read More
Answer

I'm very sorry for the delay in replying to you. I have now received feedback from the laboratory that tested the antibody ab21236 and was informed that it was tested against recombinant Epac 1 and gave a very good strong band. However, it had poor affinity for epac 1 protein when it was tagged with a marker protein (Myc-tag). Similar results were obtained with His-tag. We believe this also depend upon the orientation of the tag marker. We are not aware of a suitable positive control of endogenous Epac1 but can provide in our catalogue the recombinant protein used to test this antibody. Please let me know if you would be interested and I will add this to the catalogue. We would recommend to try to immunoprecipitate the cell extract form 250ug-500ug total protein to enrich the sample and then do a western as I have been informed that this antibody does immunoprecipitate well. Furthermore, we recommend to incubate the primary antibody overnight at 4C and to boil the samples at 95C to make sure they are well denatured. Please make sure you are using reducing conditions in the loading buffer and that your protease inhibitors are fresh. Finally, the blocking buffer sometimes can alter the signal, it would therefore be good to try a different blocking buffer, milk or BSA (typically we use 5%BSA for 1 hour and incubate the antibodies in TBST only). I hope this information will help you, please do not hesitate to contact me if I can be of further assistance,

Read More
Application
Western blot
Sample
Mouse Cell lysate - whole cell (endothelial cells, and liver)
Loading amount
10 µg
Specification
endothelial cells, and liver
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 3

Abcam user community

Verified customer

Submitted Feb 02 2007

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

Sign up