Overview

  • Product nameAnti-Calsequestrin antibody
    See all Calsequestrin primary antibodies
  • Description
    Rabbit polyclonal to Calsequestrin
  • Tested applicationsICC/IF, ICC, IP, WB, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Sheep, Rabbit, Dog, Human, Pig, Rainbow Trout
  • Immunogen

    Other Immunogen Type corresponding to Dog Calsequestrin. The immunogen is purified canine cardiac calsequestrin.

Properties

Applications

Our Abpromise guarantee covers the use of ab3516 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
ICC/IF Use at an assay dependent concentration.
ICC Use at an assay dependent concentration.
IP Use at an assay dependent concentration.
WB 1/1000 - 1/10000.

This antibody detects an ~55 kDa protein representing Calsequestrin from canine cardiac extract. Additional bands at 97 kDa may be observed and have been reported to be Calsequestrin-like proteins.

IHC-P 1/100 - 1/500.

Target

Anti-Calsequestrin antibody images

  • Immunocytochemistry/immunofluorescence analysis of C2C12 cells labeling Calsequestrin (green) with ab3516 at 1/100. Cells were ficed woth formalin and permeabilized with 0.1% Triton C-100 in TBS for 5-10 minutes and blocked with 3% BSA in PBS for 30 minutes at room temperature. Cells were incubated with the primary antibody overnight at 4°C. A DyLight-conjugated secondary antibody was used. F-actin (red) was strained with phalloidin and nuclei (blue) were stained with Hoechst or DAPI. 60X Magnification. Left - negative control.

  • ab3516 labelling Calsequestrin in the cytoplasm of Human skeletal muscle tissue (right) compared with a negative control (left) by Immunohistochemistry (formalin/PFA-fixed paraffin-embedded sections). To expose target proteins, antigen retrieval method was performed using 10mM sodium citrate (pH 6.0) microwaved for 8-15 min. Tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS. Tissue sections were incubated with primary antibody (1:200 in 3% BSA-PBS) overnight at 4°C. A HRP-conjugated anti-rabbit was used as the secondary antibody, followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

  • ab3516 labelling Calsequestrin in the cytoplasm of Mouse heart tissue (right) compared with a negative control (left) by Immunohistochemistry (formalin/PFA-fixed paraffin-embedded sections). To expose target proteins, antigen retrieval method was performed using 10mM sodium citrate (pH 6.0) microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS. Tissue sections were incubated with primary antibody (1:200 in 3% BSA-PBS) overnight at 4°C. A HRP-conjugated anti-rabbit was used as the secondary antibody, followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

  • ab3516 labelling Calsequestrin in the cytoplasm of Human heart tissue (right) compared with a negative control (left) by Immunohistochemistry (formalin/PFA-fixed paraffin-embedded sections). To expose target proteins, antigen retrieval method was performed using 10mM sodium citrate (pH 6.0) microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS. Tissue sections were incubated with primary antibody (1:200 in 3% BSA-PBS) overnight at 4°C. A HRP-conjugated anti-rabbit was used as the secondary antibody, followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

  • ab3516 staining Calsequestrin in Mouse myocyte cell by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with formaldehyde and blocked with 10% serum for 1 hour at 25°C. Samples were incubated with primary antibody (1/50 in serum) for 18 hours at 4°C. An Alexa Fluor®488-conjugated Goat anti-rabbit IgG polyclonal (1/1000) was used as the secondary antibody.

    See Abreview

  • ab3516 (1µg/ml) staining Calsequestrin in Human skeletal muscle using an automated system (DAKO Autostainer Plus). Using this protocol there is strong staining of discrete organelles within the cytoplasm.
    Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer EDTA pH 9.0 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.

  • All lanes : Anti-Calsequestrin antibody (ab3516) at 1/5000 dilution

    Lane 1 : RD cell lysate
    Lane 2 : L6 cell lysate
    Lane 3 : Mouse heart cell lysate

    Lysates/proteins at 25 µg per lane.


    Observed band size : 55 kDa (why is the actual band size different from the predicted?)

References for Anti-Calsequestrin antibody (ab3516)

This product has been referenced in:
  • Tzeng HP  et al. Dysferlin mediates the cytoprotective effects of TRAF2 following myocardial ischemia reperfusion injury. J Am Heart Assoc 3:e000662 (2014). WB ; Mouse . Read more (PubMed: 24572254) »
  • Torrado M  et al. Pitx2c is reactivated in the failing myocardium and stimulates myf5 expression in cultured cardiomyocytes. PLoS One 9:e90561 (2014). Read more (PubMed: 24595098) »

See all 15 Publications for this product

Product Wall

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunocytochemistry/ Immunofluorescence
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C
Sample Mouse Cell (myocyte)
Specification myocyte
Permeabilization No
Fixative Formaldehyde
Username

Abcam user community

Verified customer

Submitted Jun 14 2013

Abreviews
Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Loading amount 5 µg
Gel Running Conditions Reduced Denaturing (8)
Sample Human Tissue lysate - whole (Heart)
Specification Heart
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Jun 13 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Pig Tissue lysate - whole (heart)
Loading amount 7.5 µg
Specification heart
Gel Running Conditions Reduced Denaturing (7.5%)
Blocking step Invitrogen 00-0105 membrane blocking agent as blocking agent for 16 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 4°C
Username

Abcam user community

Verified customer

Submitted Apr 05 2013

I would recommened ab3516, it has been tested in more applications and species.

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Mouse Tissue lysate - whole (heart, ventricular tissue)
Loading amount 60 µg
Specification heart, ventricular tissue
Gel Running Conditions Reduced Denaturing (4~12%)
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Feb 02 2008

Thank you for your enquiry. This anti-Calsequestrin antibody (ab3516) was created against purified canine cardiac calsequestrin. Unfortunately, the exact epitope sequence has not been mapped at this time. I am sorry I can't be of more assistance ...

Read More

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