Anti-Calcium channel L type DHPR alpha 2 subunit antibody [20A] (ab2864)

Overview

  • Product nameAnti-Calcium channel L type DHPR alpha 2 subunit antibody [20A]
    See all Calcium channel L type DHPR alpha 2 subunit primary antibodies
  • Description
    Mouse monoclonal [20A] to Calcium channel L type DHPR alpha 2 subunit
  • Tested applicationsSuitable for: Flow Cyt, ICC/IF, ICC, IP, IHC-P, IHC-Fr, WBmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Rabbit, Chicken, Guinea pig, Human, Pig
  • Immunogen

    Full length native protein (purified) corresponding to Rabbit Calcium channel L type DHPR alpha 2 subunit.

  • Positive control
    • WB: rabbit skeletal muscle membrane preparations IHC: rabbit skeletal muscle, mouse brain, rat spinal cord
  • General notes

     

     

Properties

  • FormLiquid
  • Storage instructionsShipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • Storage bufferPreservative: 0.05% Sodium azide
  • Concentration information loading...
  • PurityAscites
  • Primary antibody notesVoltage-sensitive calcium channels mediate the entry of calcium into many types of excitable cells and thus play a key role in neurotransmitter release and excitation-contraction (E-C) coupling. The 1,4-dihydropyridines (DHPs) are synthetic organic compounds which can be used to identify the L-type calcium channels that are found in all types of vertebrate muscle, neuronal and neuroendocrine cells. The DHP receptor is part of the L-type calcium channel complex and is thought to be the voltage sensor in E-C coupling. The purified DHP receptor isolated from triads is composed of at least four subunits. The alpha-1 subunit contains the binding site for the DHPs and shows high sequence homology to the voltage gated sodium channel. The alpha-2 subunit is a large glycoprotein associated with the DHP receptor which was first described in skeletal muscle and is also found in high concentrations in other excitable tissues such as cardiac muscle and brain and in low concentrations in most other tissues studied. The other two subunits that co-purify with the DHP receptor are termed beta and gamma.
  • ClonalityMonoclonal
  • Clone number20A
  • IsotypeIgG2a
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab2864 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
Flow Cyt 1/200.
ICC/IF Use at an assay dependent concentration.
ICC 1/250.
IP Use at an assay dependent concentration.
IHC-P 1/500. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
IHC-Fr 1/500.
WB 1/500. Detects a band of approximately 150 kDa (predicted molecular weight: 123 kDa).

Target

  • FunctionThe alpha-2/delta subunit of voltage-dependent calcium channels regulates calcium current density and activation/inactivation kinetics of the calcium channel. Plays an important role in excitation-contraction coupling.
  • Tissue specificityIsoform 1 is expressed in skeletal muscle. Isoform 2 is expressed in the central nervous system. Isoform 2, isoform 4 and isoform 5 are expressed in neuroblastoma cells. Isoform 3, isoform 4 and isoform 5 are expressed in the aorta.
  • Sequence similaritiesBelongs to the calcium channel subunit alpha-2/delta family.
    Contains 1 cache domain.
    Contains 1 VWFA domain.
  • DomainThe MIDAS-like motif in the VWFA domain binds divalent metal cations and is required to promote trafficking of the alpha-1 (CACNA1) subunit to the plasma membrane by an integrin-like switch.
  • Post-translational
    modifications
    Proteolytically processed into subunits alpha-2-1 and delta-1 that are disulfide-linked.
  • Cellular localizationMembrane.
  • Information by UniProt
  • Database links
  • Alternative names
    • CA2D1_HUMAN antibody
    • CACN A2 antibody
    • CACNA2 antibody
    • Cacna2d1 antibody
    • CACNL2A antibody
    • Calcium channel L type alpha 2 polypeptide antibody
    • Calcium channel voltage dependent alpha 2/delta subunit 1 antibody
    • CCHL2A antibody
    • Dihydropyridine receptor alpha 2 subunit antibody
    • Dihydropyridine sensitive L type calcium channel alpha 2/delta subunit antibody
    • Dihydropyridine sensitive L type calcium channel subunits alpha 2/delta antibody
    • L type calcium channel subunit alpha 2 antibody
    • MHS 3 antibody
    • MHS3 antibody
    • Voltage dependent calcium channel subunit alpha 2/delta 1 antibody
    • Voltage gated calcium channel subunit alpha 2/delta 1 antibody
    • Voltage-dependent calcium channel subunit delta-1 antibody
    • Voltage-gated calcium channel subunit alpha-2/delta-1 antibody
    see all

Anti-Calcium channel L type DHPR alpha 2 subunit antibody [20A] images

  • Flow cytometry analysis of Dihydropyridine Receptor alpha 2 showing positive staining in the membrane and cytoplasm of SH-SY5Y cells compared to an isotype control (blue). Cells were harvested and adjusted to a concentration of 1-5x10^6 cells/ml. Cells were then fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with ab2864 at 1:100 for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.
  • Flow cytometry analysis of Dihydropyridine Receptor alpha 2 showing positive staining in the membrane and cytoplasm of Neuro-2a cells compared to an isotype control (blue). Cells were harvested and adjusted to a concentration of 1-5x10^6 cells/ml. Cells were then fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with a2864 at 1:100 for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.
  • Flow cytometry analysis of Dihydropyridine Receptor alpha 2 showing positive staining in the membrane and cytoplasm of C6 cells compared to an isotype control (blue). Cells were harvested and adjusted to a concentration of 1-5x10^6 cells/ml. Cells were then fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with ab2864 at 1:100 for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.
  • Immunofluorescent analysis of Calcium channel L type DHPR alpha 2 subunit in U251 Cells. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with a Calcium channel L type DHPR alpha 2 subunit monoclonal antibody (ab2864) at a dilution of 1:100 overnight at 4 C and incubated with a DyLight-488 conjugated secondary antibody. Calcium channel L type DHPR alpha 2 subunit staining (green) F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Images were taken at 60X magnification.
  • Immunofluorescent analysis of Calcium channel L type DHPR alpha 2 subunit in PC12 Cells. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with a Calcium channel L type DHPR alpha 2 subunit monoclonal antibody (ab2864) at a dilution of 1:20 overnight at 4 Cand incubated with a DyLight-488 conjugated secondary antibody. Calcium channel L type DHPR alpha 2 subunit staining (green) F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Images were taken at 60X magnification.
  • Immunofluorescent analysis of Calcium channel L type DHPR alpha 2 subunit in HeLa Cells. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with a Calcium channel L type DHPR alpha 2 subunit monoclonal antibody (ab2864) at a dilution of 1:100 overnight at 4 C and incubated with a DyLight-488 conjugated secondary antibody. Calcium channel L type DHPR alpha 2 subunit staining (green) F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Images were taken at 60X magnification.
  • Immunohistochemistry was performed on normal biopsies of deparaffinized mouse skeletal muscle tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:200 with a Mouse Monoclonal Antibody recognizing Calcium channel L type DHPR alpha 2 subunit (ab2864) or without primary antibody (negative control) overnight at 4ºC in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

  • Immunohistochemistry was performed on normal biopsies of deparaffinized human skeletal muscle tissue. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer and microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:100 with a Mouse Monoclonal Antibody recognizing Calcium channel L type DHPR alpha 2 subunit (ab2864) or without primary antibody (negative control) overnight at 4�C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.
  • ab2864 at 1/250 staining mouse brain tissue sections by IHC (Formalin/PFA-fixed paraffin-embedded sections). The tissue was formaldehyde fixed and a heat mediated antigen retrieval step was performed. The tissue was incubated with the antibody for 16 hours. A biotinylated goat polyclonal antibody was used as the secondary.

    See Abreview

  • ab2864 at 1/500 staining rat spinal cord tissue sections by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). The tissue was formaldehyde fixed and a heat mediated antigen retrieval step was performed, prior to incubation with the antibody for 16 hours. A biotinylated goat polyclonal antibody was used as the secondary.

    See Abreview

  • ICC/IF image of ab2864 stained PC12 cells. The cells were 4% PFA fixed (10 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 (ab2864, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti- mouse 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.

  • All lanes : Anti-Calcium channel L type DHPR alpha 2 subunit antibody [20A] (ab2864) at 1/1000 dilution

    Lane 1 : U87-MG cell lysate
    Lane 2 : Mouse brain cell lysate

    Lysates/proteins at 25 µg per lane.


    Predicted band size : 123 kDa
    Observed band size : 143 kDa (why is the actual band size different from the predicted?)
  • All lanes : Anti-Calcium channel L type DHPR alpha 2 subunit antibody [20A] (ab2864) at 1/500 dilution

    Lane 1 : Spinal Cord (Rat) Tissue Lysate
    Lane 2 : SHSY-5Y (Human neuroblastoma cell line) Whole Cell Lysate
    Lane 3 : SK N BE (Human neuroblastoma) Whole Cell Lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/5000 dilution
    developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 123 kDa
    Observed band size : 150 kDa (why is the actual band size different from the predicted?)
    Additional bands at : 18 kDa,50 kDa. We are unsure as to the identity of these extra bands.

    Exposure time : 8 minutes
    Calcium channel L type DHPR alpha 2 subunit contains a number of potential glycosylation sites (SwissProt) which may explain its migration at a higher molecular weight than predicted.
  • Overlay histogram showing SH-SY5Y cells stained with ab2864 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab2864, 1/200 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

  • IHC-Fr image of anti-Calcium channel L type DHPR alpha 2 subunit staining with ab2864 on tissue sections from chicken hindbrain. The sections were blocked with 3% BSA for 1 hour at 4°C, before incubation with ab2864 (1/1000 dilution) for 16 hours at 4°C. The secondary was an Alexa-Fluor 488 conjugated goat anti-rabbit polyclonal, used at a 1/1000 dilution.

    See Abreview

References for Anti-Calcium channel L type DHPR alpha 2 subunit antibody [20A] (ab2864)

This product has been referenced in:
  • Yamada T  et al. Nitrosative modifications of the Ca2+ release complex and actin underlie arthritis-induced muscle weakness. Ann Rheum Dis N/A:N/A (2014). WB ; Human . Read more (PubMed: 24854355) »
  • Bradley H  et al. Quantitative immunofluorescence microscopy of subcellular GLUT4 distribution in human skeletal muscle: effects of endurance and sprint interval training. Physiol Rep 2:N/A (2014). IHC-Fr ; Human . Read more (PubMed: 25052490) »

See all 11 Publications for this product

Product Wall

Application Immunohistochemistry (Frozen sections)
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 4°C
Sample Chicken Tissue sections (Hindbrain section)
Specification Hindbrain section
Permeabilization Yes - Triton X (0.1%)
Fixative Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Oct 31 2013

Thank you for your enquiry and interest.

This is a Mouse monoclonal [20A] to Calcium channel L type DHPR alpha 2 subunit so the host species of this primary antibody is mouse. The isotype is IgG2a so the compatible secondary antibody should r...

Read More

Thank you for contacting us.


No epitope mapping has been completed for this product. The immunogen is rabbit dihydropyridine receptor. We do know that this product shows positive signal in Western Blot for mouse brain and IHC for rat ...

Read More

Thank you for contacting us. Epitope mapping has not been performed for the monoclonal antibodies ab2864 and ab88486. The other antibodies are polyclonals and will bind at many different sites of the protein. Please let me know if I can be of further a...

Read More

Thank you for calling Abcam earlier today.

The antibody, ab2864was made to a native protein, and therefore, it is not available as a synthetic blocking peptide. We unfortunately do not have any of the protein available to provide either.
Read More

Thank you for contacting Abcam Technical Team and for taking the time to provide some useful details of the experiments. I am very sorry to hear that your customer is having problems with this product. Though you have kindly provided some detail...

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample Rat Tissue sections (spinal cord)
Specification spinal cord
Fixative Formaldehyde
Antigen retrieval step Heat mediated
Blocking step BSA as blocking agent for 10 minute(s) · Concentration: 1%
Username

Mr. Carl Hobbs

Verified customer

Submitted Mar 01 2007

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample Mouse Tissue sections (brain)
Specification brain
Fixative Formaldehyde
Antigen retrieval step Heat mediated
Blocking step BSA as blocking agent for 10 minute(s) · Concentration: 1%
Username

Mr. Carl Hobbs

Verified customer

Submitted Mar 01 2007

Thank you for your enquiry. Ab2864 detects 1,4 dihydropyridine (DHP) receptor alpha 2 subunit from human, rat, mouse, guinea pig and rabbit skeletal and cardiac muscle. To our knowledge, this antibody has yet to be tested in IHC on paraffin-emb...

Read More

Please find the answers to each of your three questions below: (1) We are not aware of this antibody having been used on paraformaldehyde fixed tissues. As you have no doubt seen from the datasheet, it has been used in IHC on frozen sections. (2) I ...

Read More

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