Ab37259

Manufactured by Abcam

Ab37259 is a laboratory equipment product offered by Abcam. It is designed for a specific scientific application, but a detailed description cannot be provided while maintaining an unbiased and factual approach. More information may be available from the manufacturer.

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Lab products found in correlation

Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Recombinant mouse m csf, by R&D Systems (1 mentions) Trap staining kit, by Merck Group (1 mentions) Dimethyl sulfoxide (dmso), by Merck Group (1 mentions) Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Sc 514, by Merck Group (1 mentions) Alpha modification of eagle s medium α mem, by Thermo Fisher Scientific (1 mentions) Ab2796, by Abcam (1 mentions) β tubulin, by Cell Signaling Technology (1 mentions) Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions) Sc 376875, by Santa Cruz Biotechnology (1 mentions) Phosphorylated gsk3β p gsk3β, by Cell Signaling Technology (1 mentions) Mab3026, by Merck Group (1 mentions) Nfatc1, by Cell Signaling Technology (1 mentions) Rankl, by R&D Systems (1 mentions) Catch and release v2.0 reversible immunoprecipitation system, by Merck Group (1 mentions) Ab18822, by Abcam (1 mentions) Ab30443, by Abcam (1 mentions) Ab6313, by Abcam (1 mentions) Axiovision 4, by Zeiss (1 mentions)

4 protocols using ab37259

Osteoclastogenesis Regulation by Stachydrine

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Stachydrine (purity, ≥98%) and SC79 (purity, ≥97%) were purchased from Selleck Chemicals. Alpha modification of Eagle's medium (α‐MEM), penicillin/streptomycin and foetal bovine serum (FBS) were obtained from Gibco‐BRL. Recombinant mouse M‐CSF and RANKL were purchased from R&D Systems. Primary antibodies against TAK1 (#5206), phosphorylated TAK1 (p‐TAK1) (#4508), IκBα (#4814), phosphorylated IκBα (p‐IκBα) (#2859), p65 (#8242), phosphorylated p65 (p‐p65) (#3033), IκB kinase β (IKKβ) (#8943), phosphorylated IKKα/β (p‐IKKα/β) (#2697), p38 (#9212), phosphorylated p38 (p‐p38) (#4511), ERK (#4695), phosphorylated ERK (p‐ERK) (#4370), JNK (#9252), phosphorylated JNK (p‐JNK) (#4668), phosphorylated Akt (p‐Akt) (#2965), Akt (#4685), phosphorylated GSK3β (p‐GSK3β) (#9323), GSK3β (#9315), NFATc1 (#8032), PCNA (#13110) and β‐tubulin (#2146) were obtained from Cell Signaling Technology. Primary antibodies against c‐Fos (ab208942), NFATc1 (ab2796) and CTSK (ab37259) were obtained from Abcam. Primary antibody against TRAP (sc‐376875) was obtained from Santa Cruz Biotechnology and against active p65 (MAB3026) was obtained from Millipore. Cell counting kit‐8 (CCK‐8) was obtained from Dojindo Molecular Technology. TRAP staining kit, DMSO, SC‐514 and other reagents were obtained from Sigma‐Aldrich, unless otherwise indicated.

Meng J., Zhou C., Zhang W., Wang W., He B., Hu B., Jiang G., Wang Y., Hong J., Li S., He J., Yan S, & Yan W. (2019). Stachydrine prevents LPS‐induced bone loss by inhibiting osteoclastogenesis via NF‐κB and Akt signalling. Journal of Cellular and Molecular Medicine, 23(10), 6730-6743.

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Quantifying Cathepsins and Cystatin C in AAA

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To determine the levels of cathepsins B, D, K, L and S and cystatin C in the serum of AAA patients and the 10 healthy volunteers, immunoprecipitation was performed using the Catch and Release v2.0 Reversible Immunoprecipitation System, according to the manufacturer's instructions (Millipore Corporation, Billerica, MA, USA). The following Abs were used for detection: cathepsin B (ab30443, Abcam), cathepsin D (ab6313, Abcam), cathepsin K (ab37259, Abcam), cathepsin L (ab49984, Abcam), cathepsin S (ab18822, Abcam) and cystatin C (rabbit polyclonal to cystatin C; ab33487, Abcam). Following immunoprecipitation, the samples underwent western blot analysis as described above.

Lohoefer F., Reeps C., Lipp C., Rudelius M., Haertl F., Matevossian E., Zernecke A., Eckstein H.H, & Pelisek J. (2014). Quantitative expression and localization of cysteine and aspartic proteases in human abdominal aortic aneurysms. Experimental & Molecular Medicine, 46(5), e95-.

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Osteoclast Characterization by Immunofluorescence

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Mature osteoclasts were fixed in ice-cold methanol or PBS containing 4% paraformaldehyde and stained with anti-vATPase subunit e1 (1:1000, PA5-29899, Thermo Fisher), anti-Cathepsin K (1:100, ab37259, Abcam), anti-Vinculin (1:100, V9264, Sigma), or control antibody (1:100, 5415 S, Cell Signaling), followed by fluorescently labeled secondary antibodies, following manufacturer’s instructions (Fisher). Some cells were also stained with Alexa Fluor 647-labeled Phalloidin (Thermo Fisher), following manufacturer’s instructions. The nuclei were stained with 1 µg/ml of Hoechst 33342 (Fisher) in PBS. Images were taken on an EVOS FL Auto (Thermo Fisher) and analyzed using the accompanying software. Because of intense TRAP staining in Elmo1^−/− osteoclast cultures, we counted the number of osteoclasts after cathepsin K staining by immunofluorescence. Cathepsin K-positive cells with three or more nuclei were counted using Image J. RosaYFP and Phalloidin stained osteoclast cultures were imaged on a Zeiss Imager Z2 with Apotome and analyzed using the AxioVision 4.8 software (Zeiss).

Arandjelovic S., Perry J.S., Zhou M., Ceroi A., Smirnov I., Walk S.F., Shankman L.S., Cambré I., Onengut-Gumuscu S., Elewaut D., Conrads T.P, & Ravichandran K.S. (2021). ELMO1 signaling is a promoter of osteoclast function and bone loss. Nature Communications, 12, 4974.

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Western Blot Analysis of Osteoclast Markers

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Total protein was extracted with the RIPA buffer (Beyotime, China). Then, the concentration of these samples was determined with the BCA method (Beyotime, China). After that, these proteins were separated with the 8%-12% SDS-PAGE gel (Beyotime, China). These proteins were then transferred to the PVDF membranes (Millipore, USA). Then, these membranes were blocked with 5% skim milk powder. Following that, these membranes were incubated with primary antibodies at 4°C overnight. The primary antibodies used in this study were MYC (1:500; Abcam, ab32072), c-Fos (1:500; Abcam, ab222699), nuclear factor of activated T cell (NFATc1, 1:500; Abcam, ab25916), tartrate-resistant acid phosphatase (TRAP, 1:600; Abcam, ab52750), cathepsin K (CTSK, 1:600, Abcam, ab37259), PTEN (1:600, Abcam, ab267787) and GAPDH (1:1000, Abcam, ab9485). These membranes were incubated with the horseradish peroxidase-conjugated secondary antibody (1:1000; Cell signaling technology, 7074) for 2 h on the second day. Finally, the immunoreactive signals were detected by the Pierce Western blotting Substrate (Thermo Fisher Scientific, USA).

Chen H., Li S., Yin H., Hua Z., Shao Y., Wei J, & Wang J. (2021). MYC-mediated miR-320a affects receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast formation by regulating phosphatase and tensin homolog (PTEN). Bioengineered, 12(2), 12677-12687.

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