Recombinant Anti-Caldesmon/CDM antibody [E89] KO Tested (ab32330)

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Western blot - Anti-Caldesmon/CDM antibody [E89] (ab32330)

Key features and details

Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
Rabbit monoclonal [E89] to Caldesmon/CDM
Suitable for: WB, IHC-P, Flow Cyt (Intra), IP, ICC/IF
Knockout validated
Reacts with: Mouse, Rat, Human

Alexa Fluor® 488 Alexa Fluor® 555 Alexa Fluor® 647 Carrier Free HRP PE

Product name

Anti-Caldesmon/CDM antibody [E89]
See all Caldesmon/CDM primary antibodies
Description

Rabbit monoclonal [E89] to Caldesmon/CDM
Host species

Rabbit
Tested applications

Suitable for: WB, IHC-P, Flow Cyt (Intra), IP, ICC/IFmore details
Species reactivity

Reacts with: Mouse, Rat, Human
Immunogen

Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
Positive control
- WB: HeLa and NIH/3T3 cell lysates; IHC-P: Human breast carcinoma, Human leiomyoma and mouse kidney tissue; ICC/IF: NIH/3T3 cells; IP: NIH/3T3 whole cell lysate; Flow Cyt (Intra): NIH/3T3 cells. WB: HeLa, NIH/3T3 whole cell lysates. Rat Liver lysate.
General notes
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
For more information see here.
Our RabMAb^® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb^® patents.
We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

Form

Liquid
Storage instructions

Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.
Dissociation constant (K_D)

K_D = 1.25 x 10 ^-10 M
Learn more about K_D
Storage buffer

pH: 7.20
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Concentration information loading...
Purity

Protein A purified
Clonality

Monoclonal
Clone number

E89
Isotype

IgG
Research areas

Alternative Versions
Compatible Secondaries
- Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed (ab96899)
Isotype control
- Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
KO cell lines
- Human CALD1 (Caldesmon/CDM) knockout HeLa cell line (ab265026)
KO cell lysates
- Human CALD1 (Caldesmon/CDM) knockout HeLa cell lysate (ab257375)
Positive Controls
- HeLa whole cell lysate (ab29545)
- NIH/3T3 whole cell lysate (ab7179)
Recombinant Protein
- Recombinant Human Caldesmon/CDM protein (ab114302)
Substrate reagent
- VeriBlot for IP Detection Reagent (HRP) (ab131366)

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab32330 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		1/10000 - 1/20000. Detects a band of approximately 70 kDa (predicted molecular weight: 93 kDa).
IHC-P		1/100. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Flow Cyt (Intra)		1/20. ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
IP		1/20.
ICC/IF		1/50. For unpurified use at 1/250 - 1/500.

Notes
WB 1/10000 - 1/20000. Detects a band of approximately 70 kDa (predicted molecular weight: 93 kDa).
IHC-P 1/100. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Flow Cyt (Intra) 1/20. ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
IP 1/20.
ICC/IF 1/50. For unpurified use at 1/250 - 1/500.

Function

Actin- and myosin-binding protein implicated in the regulation of actomyosin interactions in smooth muscle and nonmuscle cells (could act as a bridge between myosin and actin filaments). Stimulates actin binding of tropomyosin which increases the stabilization of actin filament structure. In muscle tissues, inhibits the actomyosin ATPase by binding to F-actin. This inhibition is attenuated by calcium-calmodulin and is potentiated by tropomyosin. Interacts with actin, myosin, two molecules of tropomyosin and with calmodulin. Also play an essential role during cellular mitosis and receptor capping.
Tissue specificity

High-molecular-weight caldesmon (isoform 1) is predominantly expressed in smooth muscles, whereas low-molecular-weight caldesmon (isoforms 2, 3, 4 and 5) are widely distributed in non-muscle tissues and cells. Not expressed in skeletal muscle or heart.
Sequence similarities

Belongs to the caldesmon family.
Domain

The N-terminal part seems to be a myosin/calmodulin-binding domain, and the C-terminal a tropomyosin/actin/calmodulin-binding domain. These two domains are separated by a central helical region in the smooth-muscle form.
Post-translational
modifications

In non-muscle cells, phosphorylation by CDK1 during mitosis causes caldesmon to dissociate from microfilaments. Phosphorylation reduces caldesmon binding to actin, myosin, and calmodulin as well as its inhibition of actomyosin ATPase activity. Phosphorylation also occurs in both quiescent and dividing smooth muscle cells with similar effects on the interaction with actin and calmodulin and on microfilaments reorganization.
Cellular localization

Cytoplasm > cytoskeleton. Cytoplasm > myofibril. On thin filaments in smooth muscle and on stress fibers in fibroblasts (nonmuscle).
Target information above from: UniProt accession Q05682 The UniProt Consortium
The Universal Protein Resource (UniProt) in 2010
Nucleic Acids Res. 38:D142-D148 (2010) .

Information by UniProt
Database links
- Entrez Gene: 800 Human
- Entrez Gene: 109624 Mouse
- Entrez Gene: 25687 Rat
- Omim: 114213 Human
- SwissProt: Q05682 Human
- SwissProt: Q62736 Rat
- Unigene: 490203 Human
Alternative names
- CAD antibody
- CALD 1 antibody
- CALD1 antibody
- CALD1_HUMAN antibody
- Caldesmon 1 antibody
- Caldesmon 1 Isoform 1 antibody
- Caldesmon 1 Isoform 2 antibody
- Caldesmon 1 Isoform 3 antibody
- Caldesmon 1 Isoform 4 antibody
- Caldesmon 1 Isoform 5 antibody
- Caldesmon antibody
- Caldesmon1 antibody
- CDM antibody
- H CAD antibody
- HCAD antibody
- L CAD antibody
- LCAD antibody
- MGC21352 antibody
- NAG22 antibody
see all

Western blot - Anti-Caldesmon/CDM antibody [E89] (ab32330)

All lanes : Anti-Caldesmon/CDM antibody [E89] (ab32330) at 1/10000 dilution (Purified)

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate
Lane 3 : Rat liver lysate

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 93 kDa
Western blot - Anti-Caldesmon/CDM antibody [E89] (ab32330)

All lanes : Anti-Caldesmon/CDM antibody [E89] (ab32330) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : CALD1 knockout HeLa cell lysate
Lane 3 : NIH/3T3 cell lysate
Lane 4 : HEK-293 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 93 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab32330 observed at 75 kDa. Red - loading control ab8245 observed at 36 kDa.

ab32330 Anti-Caldesmon/CDM antibody [E89] was shown to specifically react with Caldesmon/CDM in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265026 (knockout cell lysate ab257375) was used. Wild-type and Caldesmon/CDM knockout samples were subjected to SDS-PAGE. ab32330 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caldesmon/CDM antibody [E89] (ab32330)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human leiomyoma tissue sections labeling Caldesmon/CDM with purified ab32330 at 1/100 dilution (1.21 µg/mL). Heat mediated antigen retrieval was performed using Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used at 1/0 dilution. PBS instead of the primary antibody was used as the negative control.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument
Immunocytochemistry/ Immunofluorescence - Anti-Caldesmon/CDM antibody [E89] (ab32330)

Immunocytochemistry analysis of NIH/3T3 (Mouse embryonic fibroblast) cells labeling Caldesmon/CDM with purified ab32330 at 1/50 dilution (2.4 µg/mL). Cells were fixed in 100% Methanol and permeabilized with 0.1% TritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 µg/mL). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/mL) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.
Immunoprecipitation - Anti-Caldesmon/CDM antibody [E89] (ab32330)

Purified ab32330 at 1/20 dilution (0.6µg) immunoprecipitating Caldesmon/CDM in NIH/3T3 whole cell lysate.
Lane 1 (input): NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate.
Lane 2 (+): ab32330 + NIH/3T3 whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32330 in NIH/3T3 whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caldesmon/CDM antibody [E89] (ab32330)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse kidney tissue sections labeling Caldesmon/CDM with purified ab32330 at 1/100 dilution (1.21 µg/mL). Heat mediated antigen retrieval was performed using Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used at 1/0 dilution. PBS instead of the primary antibody was used as the negative control.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caldesmon/CDM antibody [E89] (ab32330)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat stomach tissue sections labeling Caldesmon/CDM with purified ab32330 at 1/100 dilution (1.21 µg/mL). Heat mediated antigen retrieval was performed using Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used at 1/0 dilution. PBS instead of the primary antibody was used as the negative control.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument
Flow Cytometry (Intracellular) - Anti-Caldesmon/CDM antibody [E89] (ab32330)

Intracellular Flow Cytometry analysis of NIH/3T3 (Mouse embryonic fibroblast) cells labelling Caldesmon/CDM with purified ab32330 at 1/20 dilution (10 µg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabelled control - Cell without incubation with primary antibody and secondary antibody (Blue).
Anti-Caldesmon/CDM antibody [E89] (ab32330)

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

SDS download

Country/Region

Language
Datasheet download

Download

Publishing research using ab32330? Please let us know so that we can cite the reference in this datasheet.

ab32330 has been referenced in 24 publications.

Lee LL et al. Cardiac pericytes function as key vasoactive cells to regulate homeostasis and disease. FEBS Open Bio 11:207-225 (2021). PubMed: 33135334
Du Y et al. The cancer-associated fibroblasts related gene CALD1 is a prognostic biomarker and correlated with immune infiltration in bladder cancer. Cancer Cell Int 21:283 (2021). PubMed: 34051818
Fardoun M et al. 7-O-methylpunctatin, a Novel Homoisoflavonoid, Inhibits Phenotypic Switch of Human Arteriolar Smooth Muscle Cells. Biomolecules 9:N/A (2019). PubMed: 31717401
Nakagaki-Silva EE et al. Identification of RBPMS as a mammalian smooth muscle master splicing regulator via proximity of its gene with super-enhancers. Elife 8:N/A (2019). PubMed: 31283468
Domke LM & Franke WW The cell-cell junctions of mammalian testes: II. The lamellar smooth muscle monolayer cells of the peritubular wall are laterally connected by vertical adherens junctions-a novel architectonic cell-cell junction system. Cell Tissue Res N/A:N/A (2018). PubMed: 30591979

View all Publications for this product

Customer reviews and Q&As

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1-6 of 6 Abreviews or Q&A

Question

Thank you for your kind reply.

The customer has also requested some specific instructions or a more precise protocol regarding incubation times and recommended buffers for FACS and IF.

Also, could you please mention a cell type or cell line that might serve as a good negative control for Caldsmon?

Any cells known not to express Cladesmon might do.

Thank you for your kind support.

Regards,

Abcam community

Verified customer

Asked on Feb 20 2013

Answer

I have attached an image of our Flow cytometry results with ab32330.

We used untreated HeLa cells as our negative control and Anisomycin-treated HeLa cells as our positive control. The primary dilution was 1:20. Both were permeabilized according to our standard protocol which is described below:

1. Solutions and Reagents
1.1. 1X PBS

1.2. Blocking buffer: 0.5% BSA in 1X PBS

1.3. 2% paraformaldehyde (1% solution - optional for storing samples)

1.4. 1X FACS permeabilizing solution (BD Biosciences cat. #340973)

1.5. Fluorescently-conjugated secondary antibody (various forms)

2. Protocol
2.1. Collect 1x106 cells/sample.

2.2. Wash cells once with blocking buffer.

2.3. Fix cells with 2% paraformaldehyde and incubate at room temperature for 10 min.

2.4. Wash cells once with blocking buffer.

2.5. Add 0.5 ml 1X FACS permeabilizing solution and incubate at room temperature for 10 min.

2.6. Wash cells once with blocking buffer.

2.7. Incubate cells in blocking buffer for 30 min at room temperature.

2.8. Add primary antibody at the appropriate dilution and incubate for 30 min at room temperature.

2.9. Wash twice with blocking buffer and incubate with fluorescently-conjugated secondary antibody for 30 min at room temperature.

2.10. Wash cells twice with blocking buffer.

2.11. Re-suspend cells in 1X PBS and analyze on flow cytometry. Samples can be kept in 1% paraformaldehyde at 4 °C overnight.

Please let me know if you have any additional questions.

Abcam Scientific Support

Answered on Feb 20 2013

Question

Dear Tech support

Could you please specify what is the formal shelf life of product ab32330?
I know it's guaranteed for 6 month from delivery but how long is good for when stored properly?

Thank you for your kind advise.
Regards,

Abcam community

Verified customer

Asked on Jan 03 2013

Answer

Thank you for your enquiry regarding ab32330.

Generally, antibodies can be stored at -20°C for several years if the storage buffer contains cryoprotective agent and the aliquot size is no smaller than 10 µl. Please always check the product datasheet for storage information.

However, I would like to emphasize that Abcam can only offer a full guarantee called Abpromise within 6 months of purchase.

https://www.abcam.com/index.html?pageconfig=resource&rid=10795

If you need any further assistance in the future, please do not hesitate to contact me.

Abcam Scientific Support

Answered on Jan 03 2013

Question

The antibody is needed for Flow Cytometry in Human or Mouse.
Thank you for your help!

Regards,

Abcam community

Verified customer

Asked on Dec 21 2012

Answer

Thank you for your reply and for kindly providing the further information.

We have one antibody for detection of Caldesmon which has been tested in flow cytometry and mouse, which I think is the one you already found:

Caldesmon antibody [E89] (ab32330)

The sequence we used to raise this antibody has 100% homology with all 6 Caldesmon isoforms. Depending on the level of expression in the customer's sample, they may be able to observe all 6.

Regrettably, this is therefore not specific for the smaller isoforms of the protein in this case and may not be what the customer is looking for.

If you have any further questions, please do not hesitate to contact me.

Abcam Scientific Support

Answered on Dec 21 2012

Question

Is antibody ab32330 caldesmon isoform1 specific? could you please specify what is the isoform specificity and if it's not isoform 1, is there an antibody for this protein?

Abcam community

Verified customer

Asked on Jul 04 2012

Answer

Thank you for contacting us.

Checking the immunogen sequence for this antibody, this antibody is not specific to Caldesmon isoform 1. Based on the sequence, it might crossreact with isoform 1-6 of human Caldesmon protein.

We have two antibodies which react only with the heavy form/ Isoform 1:

ab50016 Anti-Caldesmon antibody [hHCD]

https://www.abcam.com/CaldesmonCDM-antibody-hHCD-ab50016.html (or use the following: https://www.abcam.com/CaldesmonCDM-antibody-hHCD-ab50016.html).

and ab11013 Anti-Caldesmon antibody [CALD 5]

https://www.abcam.com/CaldesmonCDM-antibody-CALD-5-ab11013.html (or use the following: https://www.abcam.com/CaldesmonCDM-antibody-CALD-5-ab11013.html).

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

Abcam Scientific Support

Answered on Jul 04 2012

Question

I am wondering if this antibody (ab32330) recognize the high-molecular weight caldesmon? As shown on your website there are different isoforms in different tissue and I'd like to get antibody for isoform 1.

Abcam community

Verified customer

Asked on Dec 28 2011

Answer

Thank you for contacting us. Our source for this antibody confirms that the antibody does recognize a 120-150 kDa protein in smooth muscle samples. They do not, have a blot image, however. Please do not hesitate to contact us if you need any more advice or information.

Abcam Scientific Support

Answered on Dec 28 2011

Question

Dear sirs, I have ordered your product no ab 32330, rabbit monoclonal to Caldesmon. In the datasheet it says IgG as an isotype. Is this supposed to be IgG1 or what does it mean? With kind regards

Abcam community

Verified customer

Asked on Dec 19 2011

Answer

Thank you for your patience. The isotype of the anti-Caldesmon antibody is simply rabbit IgG. In immunology, no specific isotype has been determined for rabbits. It is only known for human and mouse. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Abcam Scientific Support

Answered on Dec 19 2011

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Key features and details

Related conjugates and formulations

Overview

Product name

Description

Host species

Tested applications

Species reactivity

Immunogen

Positive control

General notes

Properties

Form

Storage instructions

Dissociation constant (KD)

Storage buffer

Purity

Clonality

Clone number

Isotype

Research areas

Associated products

Alternative Versions

Compatible Secondaries

Isotype control

KO cell lines

KO cell lysates

Positive Controls

Recombinant Protein

Substrate reagent

Applications

The Abpromise guarantee

Target

Function

Tissue specificity

Sequence similarities

Domain

Post-translationalmodifications

Cellular localization

Database links

Alternative names

Images

Protocols

Datasheets and documents

SDS download

Datasheet download

References (24)

Customer reviews and Q&As

Dissociation constant (K_D)

Post-translational
modifications