Validated using a knockout cell line
Recombinant
RabMAb

Recombinant Anti-Calpain small subunit 1 antibody [EPR3324] (ab92333)

Overview

  • Product name

    Anti-Calpain small subunit 1 antibody [EPR3324]
    See all Calpain small subunit 1 primary antibodies
  • Description

    Rabbit monoclonal [EPR3324] to Calpain small subunit 1
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, IHC-P, ICC, Flow Cytmore details
    Unsuitable for: IP
  • Species reactivity

    Reacts with: Mouse, Rat, Human, Chinese hamster
  • Immunogen

    Synthetic peptide within Human Calpain small subunit 1 aa 100-200. The exact sequence is proprietary.

  • Positive control

    • WB: fetal brain or fetal spleen tissue lysate; T47D or 293T cell lysate. IHC: Human kidney tissue.
  • General notes

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab92333 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
IHC-P
ICC
Flow Cyt
  • Application notes
    Flow Cyt: 1/50.
    ICC: 1/100 - 1/250.
    IHC-P: 1/100 - 1/250. Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol. The use of an HRP/AP polymerized antibody will give a stronger signal.

    WB: 1/1000 - 1/5000. Predicted molecular weight: 28 kDa.

    Is unsuitable for IP.


    Not yet tested in other applications.
    Optimal dilutions/concentrations should be determined by the end user.
  • Target

    • Function

      Regulatory subunit of the calcium-regulated non-lysosomal thiol-protease which catalyzes limited proteolysis of substrates involved in cytoskeletal remodeling and signal transduction.
    • Sequence similarities

      Contains 5 EF-hand domains.
    • Domain

      The contact of the 5th EF-hand domain from each monomer allows the formation of the homodimer and also appears to mediate the contact between the large catalytic subunit and small regulatory subunit for the formation of the heterodimer.
      EF-hand domains are paired. EF-hand 1 is paired with EF-hand 2 and EF-hand 3 is paired with EF-hand 4. The fifth EF-hand domain, left unpaired, does not bind the calcium but is responsible of the dimerization by EF-embrace. The first four EF-hand domains bind calcium, however it is not sure if the binding of EF-hand 4 to calcium is physiologically relevant.
    • Cellular localization

      Cytoplasm. Cell membrane. Translocates to the plasma membrane upon calcium binding.
    • Information by UniProt
    • Database links

    • Alternative names

      • 30K antibody
      • Calcium activated neutral proteinase antibody
      • Calcium activated neutral proteinase small subunit antibody
      • Calcium dependent protease small subunit 1 antibody
      • Calcium dependent protease small subunit antibody
      • Calcium-activated neutral proteinase small subunit antibody
      • Calcium-dependent protease small subunit 1 antibody
      • Calcium-dependent protease small subunit antibody
      • Calpain 4 antibody
      • Calpain 4 small subunit (30K) antibody
      • Calpain 4 small subunit antibody
      • Calpain regulatory subunit antibody
      • Calpain S1 antibody
      • Calpain small polypeptide antibody
      • Calpain small subunit 1 antibody
      • Calpain4 antibody
      • CalpainS1 antibody
      • CANP antibody
      • CANP small subunit antibody
      • CANPS antibody
      • CAPN 4 antibody
      • CAPN S1 antibody
      • CAPN4 antibody
      • CAPNS 1 antibody
      • CAPNS antibody
      • Capns1 antibody
      • CDPS antibody
      • CPNS1_HUMAN antibody
      • CSS 1 antibody
      • CSS1 antibody
      see all

    Images

    • Lane 1: Wild type HAP1 whole cell lysate (20 µg)
      Lane 2: Calpain small subunit 1 knockout HAP1 whole cell lysate (20 µg)
      Lane 3: A431 whole cell lysate (20 µg)
      Lane 4: HEK293 whole cell lysate (20 µg)

       

      Lanes 1 - 4: Merged signal (red and green). Green - ab92333 observed at 28 kDa. Red - loading control, ab8245, observed at 37 kDa.

       

      ab92333 was shown to recognize Calpain small subunit 1 when Calpain small subunit 1 knockout samples were used, along with additional cross-reactive bands. Wild-type and Calpain small subunit 1 knockout samples were subjected to SDS-PAGE. Ab92333 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

    • All lanes : Anti-Calpain small subunit 1 antibody [EPR3324] (ab92333) at 1/2000 dilution

      Lane 1 : T47D
      cell lysate
      Lane 2 : fetal brain lysate
      Lane 3 : fetal spleen lysate
      Lane 4 : 293T
      cell lysate

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : Goat anti-Rabbit HRP at 1/2000 dilution

      Predicted band size: 28 kDa
      Observed band size: 28 kDa

    • Immunohistochemistry staining of Calpain small subunit 1 in formalin-fixed, paraffin-embedded Human kidney tissue using 1/100 ab92333.

      Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.

      Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.

    • Overlay histogram showing HeLa cells stained with ab92333 (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 (ab92333, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

    References

    This product has been referenced in:

    • Li FZ  et al. Crosstalk between calpain activation and TGF-ß1 augments collagen-I synthesis in pulmonary fibrosis. Biochim Biophys Acta 1852:1796-804 (2015). Read more (PubMed: 26071646) »
    • Cai JJ  et al. Increased expression of Capn4 is associated with the malignancy of human glioma. CNS Neurosci Ther 20:521-7 (2014). Read more (PubMed: 24628706) »
    See all 3 Publications for this product

    Customer reviews and Q&As

    Answer

    Thank you for taking the time to contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this lot of antibody, particularly as the previous vial has worked.

    I would like to reassure you that this antibody is tested and covered by our 6 month guarantee for WB and rat samples. In the event that a product is not functioning in the tested applications and species cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.

    I would like to investigate this particular case further for you, and also obtain more information for our quality monitoring records. In order to proceed with this, I have enclosed a technical questionnaire below. I would appreciate if you could complete this. It will help you put the information we require together very easily.

    I would appreciate if youare also able to provide any images which would help us to assess the results.

    Thank you for your time and cooperation. We look forward to receiving the completed questionaire.


    Order Details
    Antibody code:

    Problem
    Choose: Non-specific band Multiple bands No signal or weak signal High background

    Lot number

    Purchase order number
    or preferably Abcam order number:



    General Information
    Antibody storage conditions (temperature/reconstitution etc)


    Description of the problem (high background, wrong band size, more bands, no band etc.)


    Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.)


    Sample preparation (Buffer/Protease inhibitors/Heating sample etc.)


    Amount of protein loaded


    Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.)


    Transfer and blocking conditions (Buffer/time period, Blocking agent etc.)


    Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


    Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


    Detection method (ECL, ECLPlus etc.)


    Positive and negative controls used (please specify)



    Optimization attempts (problem solving)
    How many times have you tried the Western?



    Have you run a "No Primary" control?
    Yes No

    Do you obtain the same results every time?
    Yes No
    e.g. are the background bands always in the same place?


    What steps have you altered?


    Additional Notes:


    Image:
    We would appreciate if you are able to provide an image (including molecular weight markers) which would help us to asess the results.

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