Calnexin

Manufactured by Cell Signaling Technology

Sourced in United States, United Kingdom

Calnexin is a chaperone protein that assists in the folding and quality control of newly synthesized glycoproteins in the endoplasmic reticulum. It binds to and retains misfolded or incompletely assembled glycoproteins, allowing them to undergo additional folding or targeting for degradation.

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Anti-calnexin (54 citations) Rabbit anti-calnexin (16 citations) Rabbit anti-Calnexin antibody (3 citations) Calnexin antibody (3 citations) Anti-Calnexin (C5C9) (3 citations) Calnexin (C5C9) (3 citations) Calnexin (2679) (3 citations) Calnexin (C5C9) rabbit monoclonal antibody (2 citations) Anti‐Calnexin antibody (2 citations)

137 protocols using calnexin

Antibody Procurement and Inhibitor Sources

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Antibodies against GP73, MAVS, GAPDH, AIF, MBP and GST were purchased from Proteintech (Wuhan, Hubei, China). Antibody against HCV NS5A (2F6) was purchased from BioFront (Anhui, Hefei, China). Antibodies against HCV Core and p-IRF3 were purchased from Abcam (Cambridge, MA, USA). Antibody against IRF3 was purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). Antibodies against Flag, HA, c-Myc, TRAF6, Calnexin, Syntaxin 6, p-IκBα and IκBα were purchased from Cell Signaling Technology (Danvers, MA, USA). All inhibitors were purchased from Selleck Chemicals (Houston, TX, USA).

Zhang X., Zhu C., Wang T., Jiang H., Ren Y., Zhang Q., Wu K., Liu F., Liu Y, & Wu J. (2017). GP73 represses host innate immune response to promote virus replication by facilitating MAVS and TRAF6 degradation. PLoS Pathogens, 13(4), e1006321.

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Exosome Protein Characterization in Oxycodone Addiction

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Exosome markers proteins were identified by Western blotting. 20 μg of TE from both control and oxycodone SA monkeys were pooled (n = 3/group), denatured directly in 2x sample loading buffer and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 8–12% Tris-glycine gel (as required based upon the protein molecular weight). The separated proteins were transferred on to nitrocellulose membrane followed by blocking with 5% non-fat milk powder (w/v) in Tris-buffered saline (10 mM Tris–HCl, pH 7.5, 100 mM NaCl, 0.1% Tween 20) for 1 h at room temperature. Membranes were probed for the desired protein using specific primary antibodies: CD9 (Cat. No. ab92726), CD31 (Cat. No. ab28364), Alix (Cat no. ab88388) (all three from Abcam, Cambridge, Massachusetts, USA), HSP70 (Cat. No. 4872S), Calnexin (Cat no. 2679s) (both from Cell Signaling Technology, Beverly, Massachusetts, USA). This was followed by the appropriate peroxidase-conjugated secondary antibody and visualized by the ECL detection system. The autoradiograms/bands were scanned using Adobe Photoshop 6.0 (Adobe Systems, San Jose, CA).

Kumar A., Kim S., Su Y., Sharma M., Kumar P., Singh S., Lee J., Furdui C.M., Singh R., Hsu F.C., Kim J., Whitlow C.T., Nader M.A, & Deep G. (2021). Brain cell-derived exosomes in plasma serve as neurodegeneration biomarkers in male cynomolgus monkeys self-administrating oxycodone. EBioMedicine, 63, 103192.

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Immunoblot Analysis of Cell Signaling Pathways

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Immunoblot analysis was carried out as described.^{31 (link)} Primary antibodies used were: Calnexin (#2433), p-cJUN serine 73 (#3270), cJUN (#9165), ERK (#9102), pAKT serine 473 (#4060), p-S6 (#4858) (all from Cell Signaling. 1:1000), and MAP3K1 (ab220416, Abcam; 1:100). Band intensities were quantitated using ImageJ. All pictured blots derive from the same experiment and were processed in parallel.

Nixon M.J., Formisano L., Mayer I.A., Estrada M.V., González-Ericsson P.I., Isakoff S.J., Forero-Torres A., Won H., Sanders M.E., Solit D.B., Berger M.F., Cantley L.C., Winer E.P., Arteaga C.L, & Balko J.M. (2019). PIK3CA and MAP3K1 alterations imply luminal A status and are associated with clinical benefit from pan-PI3K inhibitor buparlisib and letrozole in ER+ metastatic breast cancer. NPJ Breast Cancer, 5, 31.

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Western Blot Analysis of Lipid Droplet Proteins

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Samples were mixed with 2× Laemmli buffer and subjected to 7.5% or 10% SDS-PAGE. After electrophoresis, the proteins were transferred to Hybond-C nitrocellulose filters (GE Healthcare). Incubations with primary antibodies were performed at 4°C overnight. Primary antibodies used were rabbit polyclonal to ORP5 (Sigma-Aldrich, HPA038335), GAPDH (Cell Signaling Technology, 2118), Calnexin (Cell Signaling Technology, 2433), β-actin (Cell Signaling Technology, 4970), and CGI-58/ABHD5 (Proteintech, 12201-1-AP) and mouse monoclonal to GFP (Santa Cruz Biotechnology, sc-9996). Secondary antibodies were peroxidase-conjugated AffiniPure donkey anti-rabbit or donkey anti-mouse IgG (H+L; Jackson ImmunoResearch Laboratories) used at a 1:5,000 dilution. The bound antibodies were detected by enhance chemiluminescence Western blotting detection reagent (GE Healthcare or Merck Millipore) and visualized with Molecular Imager ChemiDoc XRS+ (Bio-Rad Laboratories).

Du X., Zhou L., Aw Y.C., Mak H.Y., Xu Y., Rae J., Wang W., Zadoorian A., Hancock S.E., Osborne B., Chen X., Wu J.W., Turner N., Parton R.G., Li P, & Yang H. (2019). ORP5 localizes to ER–lipid droplet contacts and regulates the level of PI(4)P on lipid droplets. The Journal of Cell Biology, 219(1), e201905162.

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Characterization of Extracellular Vesicles

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1.5E10 particles of C-EVs and 5E10 particles of BR-EVs were prepared in Laemmli buffer under non-reducing conditions, and separated by SDS-PAGE and transferred onto PVDF membranes, and probed with antibodies diluted 1:500 against human CD9 (HBD-CD9, HansaBioMed Life Sciences Ltd), CD29 (#610,467, BD Transduction Laboratories), CD81 (HBD-CD81-EM4, HansaBioMed Life Sciences Ltd), TSG101 (BD Biosciences), and 1:1000 Calnexin (Cell Signalling Technology). Proteins of interest were detected with 1:3000 diluted HRP-conjugated IgG antibody (NA931 anti-mouse HRP, or NA934 anti-rabbit HRP, GE Healthcare) and visualized with the Clarity ECL substrate (BioRad).

Palviainen M., Saari H., Kärkkäinen O., Pekkinen J., Auriola S., Yliperttula M., Puhka M., Hanhineva K, & Siljander P.R. (2019). Metabolic signature of extracellular vesicles depends on the cell culture conditions. Journal of Extracellular Vesicles, 8(1), 1596669.

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Immunoblotting Analysis of Cellular Proteins

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Cellular protein extracts were prepared using RIPA lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% sodium deoxycholate, and 0.1% SDS). Protein lysates were subjected to immunoblotting using antibodies against SOD1, poly ADP ribose polymerase (PARP, BD Bioscience Pharmingen, San Diego, CA), caspase-3, caspase-8, p53, p21, MCL-1, Bcl_xL, c-Myc, cytochrome-c (Santa Cruz Biotechnology), caspase-9, p-eIF2α (Abcam, Cambridge, MA), cyclin-B1, CDC25C, CDC2, HSP60, CLPP, COX IV, PERK, BIP, Calnexin, GFP (Cell Signaling, Beverly, MA), polyubiquitin (Enzo Life Sciences, Inc., Farmingdale, NY), GAPDH, or β-actin (Sigma-Aldrich).

Du T., Song Y., Ray A., Chauhan D, & Anderson K.C. (2020). Proteomic analysis identifies mechanism(s) of overcoming bortezomib resistance via targeting ubiquitin receptor Rpn13. Leukemia, 35(2), 550-561.

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Extracellular Vesicle Protein Profiling

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EVs, NEEVs, EV-depleted plasma, total EV after enrichment, and cells were denatured directly in a 4X Laemmli sample loading buffer and separated by SDS-PAGE using Mini PROTEAN^® TGX^™ precast gels (Bio-Rad, Catalog # 4561044). Separated proteins were transferred unto polyvinylidene difluoride (PVDF) membranes using a Trans-Blot^® Turbo transfer system (Bio-Rad, Catalog # 1704156). Primary antibodies used include CD171 (1:1000, Invitrogen, Catalog # 13–1719-82), CD81 (1:1000, Santa Cruz, Catalog # SC-166029), Alix (1:1000, Santa Cruz, Catalog # SC-53540), and calnexin (1:1000, Cell Signaling, Catalog # 2679).

Burrows K., Figueroa-Hall L., Stewart J., Alarbi A., Kuplicki R., Hannafon B., Tan C., Risbrough V., McKinney B., Ramesh R., Victor T., Aupperle R., Savitz J., Teague K., Khalsa S, & Paulus M. (2023). Exploring the role of neuronal-enriched extracellular vesicle miR-93 and interoception in major depressive disorder. Research Square.

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PD-L1 Expression Analysis in LUAD Samples

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WB was implemented to assess PD‐L1 level in LUAD tumor tissues (which were acquired from LUAD patients before and after a 15 weeks‐treatment with PD‐L1 inhibitors), HCC827 cells transfected with NC vector or pcDNA‐PD‐L1, and xenograft tumor tissues (which were obtained from different HCC827 cells (NC vector, pcDNA‐PD‐L1, pcDNA‐PD‐L1 + HCC827‐miR‐16‐5p mimic‐exosome). Moreover, WB was used to identify the expression of TSG101 and CD63 (exosome superficial marker proteins) in exosomes isolated from serum samples and cell culture medium. The WB procedure was carried out exactly as previously reported.²⁴, ³² The primary antibodies utilized in this study were listed below: PD‐L1 (1:1000; Abcam), TSG101 (1:2000; Abcam), CD63 (1:1000; Proteintech), β‐actin (1:2000; Abcam), and Calnexin (1:2000; Cell Signaling Technology). β‐actin was employed as an internal control of PD‐L1 for standardized quantification, while Calnexin was utilized as an internal reference of TSG101 and CD63

Chen H., Luo Y., Lin M., Peng X., Liu M., Wang Y., Li S., Yang D, & Yang Z. (2022). Serum exosomal miR‐16‐5p functions as a tumor inhibitor and a new biomarker for PD‐L1 inhibitor‐dependent immunotherapy in lung adenocarcinoma by regulating PD‐L1 expression. Cancer Medicine, 11(13), 2627-2643.

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Western Blot Analysis of Mitochondrial Proteins

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Western blot analysis was performed, as previously described (43 (link)). The following antibodies were used: F₁F₀ ATPase (adenosine triphosphatase) (1:1000; BD, 612518), vinculin (1:2000; Santa Cruz Biotechnology, sc-55465), calnexin (1:1000; Cell Signaling Technology, 2679), Flag M2 (1/1000; Sigma-Aldrich, F3165), and HA (1:1000; Ozyme, BLE901501), cytochrome c (1:1000; BD Bioscience, 556433). Horseradish peroxidase–conjugated goat anti-mouse, rabbit anti-mouse, and goat anti-rabbit secondary antibodies (DAKO) were used as secondary antibodies. Western blot analysis was performed according to standard procedures.

Popgeorgiev N., Sa J.D., Jabbour L., Banjara S., Nguyen T.T., Akhavan-E-Sabet A., Gadet R., Ralchev N., Manon S., Hinds M.G., Osigus H.J., Schierwater B., Humbert P.O., Rimokh R., Gillet G, & Kvansakul M. (2020). Ancient and conserved functional interplay between Bcl-2 family proteins in the mitochondrial pathway of apoptosis. Science Advances, 6(40), eabc4149.

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Immunoblotting Experimental Protocol

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Immunoblotting experiments were conducted as previously described (15 (link)). Antibodies used for immunoblotting were specific for the following proteins: LC3b, PERK, IRE1α, P-AMPK, AMPK (total), P-ULK (Ser⁷⁵⁷), P-ULK (Ser³¹⁷), ULK (total), COXIV, cytochrome c, calnexin, HSP60, mitofusin-1, and Bcl-2 (Cell Signaling Technology); BAP31, ATF6, Tom40, Tom22, prohibitin, and VDAC1 (Santa Cruz Biotechnology); Parkin and FACL-4 (Abcam); and NDUFS4, NDUFB11, α-tubulin, β-actin, and flag (Sigma-Aldrich). Antibodies were diluted to 1:1000, except for NDUFS4 (1:500) or anti–β-actin (1:10,000). Secondary antibodies were purchased from Promega (anti-rabbit and anti-mouse at 1:5000).

, & Namba T. (2019). BAP31 regulates mitochondrial function via interaction with Tom40 within ER-mitochondria contact sites. Science Advances, 5(6), eaaw1386.

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