Rabbit anti calnexin

Manufactured by Cell Signaling Technology

Sourced in United States

Rabbit anti-calnexin is an antibody that specifically recognizes the calnexin protein, which is an integral membrane protein of the endoplasmic reticulum (ER) involved in the quality control of protein folding. This antibody can be used to detect and study the expression and localization of calnexin in various cell types and tissues.

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Close spelling variants of this product

Calnexin (137 citations) Anti-calnexin (54 citations) Rabbit anti-Calnexin antibody (3 citations)

16 protocols using rabbit anti calnexin

EV Protein Level Analysis via Western Blotting

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For analysis of protein levels in EVs, isolated EVs were lysed with RIPA buffer, followed by addition of 1X Laemlli buffer and incubation at 95°C for 5 min. Samples were subject to standard Western blotting with chemiluminescence detection. The following antibodies were used as recommended by the manufacturer: rabbit anti‐Src (Cat#: 2109, 1:1000), rabbit anti‐calnexin (Cat#: 2679, 1:5000), rabbit anti‐CD‐9 (Cat#: 13403 for human species, 1:1000), rabbit anti‐GAPDH (Cat#: 2118, 1:10,000), rabbit anti‐gamma‐tubulin (Cat#: 5886, 1:1000), and mouse anti‐Cas9 (Cat#: 14697, 1:1000) were all purchased from Cell Signaling Technology. Mouse anti‐VSV‐G (Cat#: EB0010, Kerafast, 1:1000), rabbit anti‐syntenin (Cat#: ab19903, Abcam, 1:1000), mouse anti‐CD63 (Cat#: 556019, BD Pharmingen, 1:500). Secondary antibodies anti‐rabbit IgG HRP (Cat#: 7074, 1:5000), anti‐mouse IgG HRP (Cat# 7076, Cell Signaling Technology, 1:5000). The anti‐myristoylated octapeptide antibody described in this study was used at dilutions of 1:250, 1:500, or 1:1000. Cas9 recombinant protein was purchased from Sigma Aldrich (Cat# Cas9Prot). EV protein analysis was done in accordance with the MISEV 2018 guidelines (Théry et al., 2018 (link)). The band intensity was quantified by Image J software.

Whitley J.A., Kim S., Lou L., Ye C., Alsaidan O.A., Sulejmani E., Cai J., Desrochers E.G., Beharry Z., Rickman C.B., Klingeborn M., Liu Y., Xie Z, & Cai H. (2022). Encapsulating Cas9 into extracellular vesicles by protein myristoylation. Journal of Extracellular Vesicles, 11(4), e12196.

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Exosome Characterization by Western Blot

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Exosome-specific markers were detected by western blot analysis (WB) [19 (link),40 (link)]. Thirty micrograms of exosomes were used to perform SDS-PAGE, followed by semidry transfer. The following primary antibodies were used (overnight incubation): rabbit anti-ALIX (Abcam), rabbit anti-Tsg101 (Abcam), rabbit anti-calnexin (Cell Signaling Technology) and mouse anti-Hsp70 (Santa Cruz Biotechnology). The following peroxidase-conjugated antibodies were used: anti-rabbit (Abcam) and anti-mouse (Merck). The membranes were incubated in ECL (Cell Signaling Technology). Proteins were visualized using a ChemiDocTM XRS + system (BioRad). Protein lysate of human adipose MSCs were used as a positive control.

Villatoro A.J., Martín-Astorga M.D., Alcoholado C., Sánchez-Martín M.D, & Becerra J. (2021). Proteomic Analysis of the Secretome and Exosomes of Feline Adipose-Derived Mesenchymal Stem Cells. Animals : an Open Access Journal from MDPI, 11(2), 295.

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Immunofluorescence Labeling Protocol

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Antibodies used were rabbit anti-Flag (Sigma), mouse anti-Flag M1 (Sigma), mouse anti-Flag M2 (Sigma), mouse anti-HA 16B12 (Biolegend), rat anti-HA (Roche), goat anti-AC9 (Santa Cruz Biotech), mouse anti-Golgin-97 (Thermo), rabbit anti-calnexin (Cell Signaling), mouse anti-Sodium/Potassium ATPase (Fisher).

Lazar A.M., Irannejad R., Baldwin T.A., Sundaram A.B., Gutkind J.S., Inoue A., Dessauer C.W, & Von Zastrow M. (2020). G protein-regulated endocytic trafficking of adenylyl cyclase type 9. eLife, 9, e58039.

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Western Blot Antibody Dilutions

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The antibodies with their dilutions and sources were as follows: Antibodies for western blotting: rabbit polyclonal anti-HA (1:4000; H6908, Sigma, Lot No: 022M4806), mouse monoclonal anti-α-tubulin (1:10,000; Sigma, T5168, Lot No: 103M4773V), goat anti-SEL1L (1: 200; Santa Cruz Biotechnology, SC-48081, Lot No: C3109), Rabbit anti-HRD1 (1:500: Cell Signaling technology, 12925 S, Lot No: 1), rabbit anti-OS-9 (1: 500: Abcam, ab19853, Lot No: GR54041-1), rabbit anti-Calnexin (1:1000; Cell Signaling Technology, 2433 S, Lot No: 2), mouse monoclonal anti-ubiquitin (1:1000; Sigma, U0508, Lot No: SLBL1928V), Rabbit anti-Histone-H3 (1:1000; Cell Signaling Technology, 9715S, Lot No: 18), Rabbit anti-GAPDH (1: 2500; Abcam, ab9485, Lot No: GR184357-1), Rabbit anti-LC3-B (1: 1000; Sigma, L7543, Lot No: 046M4787V), goat anti-rabbit IgG-peroxidase (1: 50,000; Sigma), rabbit anti-mouse IgG-peroxidase (1:80,000; Sigma), chicken anti-goat IgG-peroxidase (1:5000, Santa Cruz Biotechnology).

Kizhakkedath P., John A., Al-Gazali L, & Ali B.R. (2018). Degradation routes of trafficking-defective VLDLR mutants associated with Dysequilibrium syndrome. Scientific Reports, 8, 1583.

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Immunostaining of Eag1, Calnexin, and Cadherin

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Cells grown on coverslips were rinsed in ice-cold PBS, and then fixed for 20 min with 4% paraformaldehyde in PBS at 4 °C. After washing with cold PBS, fixed cells were permeabilized and blocked with a blocking buffer (10% normal goat serum in PBS, 0.05% (v/v) Triton X-100) for 60 min at 4 °C. Cells were then incubated with the following primary antibodies overnight at 4 °C: rabbit anti-Eag1 (1:1000; Alomone, Jerusalem, Israel), rabbit anti-calnexin (1:100; Cell signaling Technology, Danvers, MA, USA), or mouse anti-cadherin (1:100; Abcam, Cambridge, MA, USA). After several washes with PBST (PBS with 0.05% Triton X-100), cells were incubated with Alexa Fluor 488-conjugated rabbit IgG and Alexa Fluor 568-conjugated mouse IgG (1:500; Invitrogen, Carlsbad, CA, USA) secondary antibodies for one hour at room temperature. Cell nuclei were labeled with DAPI (1 μg/ml in PBS; Sigma, St. Louis, MO, USA) at room temperature. After final wash, coverslips were mounted in a mounting medium (4% n-propylgallate, 90% glycerol, 0.1 M carbonate buffer, pH 9.2), and viewed using a Zeiss LSM880 laser-scanning microscope (Zeiss, Oberkochen, Germany).

Hsieh C.H., Chou C.C., Fang Y.C., Hsu P.H., Chiu Y.H., Yang C.S., Jow G.M., Tang C.Y, & Jeng C.J. (2023). 14-3-3 proteins regulate cullin 7-mediated Eag1 degradation. Cell & Bioscience, 13, 18.

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Comprehensive Antibody Panel for Protein Analysis

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The following primary antibodies were used: Rabbit anti-PRAS40, IBL; Rabbit anti-PARP, Cell Signaling, 9542; Mouse anti-GAPDH, Chemicon/Millipore; Rabbit anti-Calnexin, Cell Signaling, 2433; Rabbit anti-RPL11, Abcam, ab79352; Rabbit anti-Raptor, Millipore, 09-217; Rabbit anti-14-3-3 epsilon, Santa Cruz, sc-1020; Rabbit anti-pan-14-3-3, Santa Cruz, sc -629; Mouse anti-HSP90, Santa Cruz, sc-13119; Mouse anti-Flag, Sigma M2; Rabbit anti-mTOR, Cell Signaling, 2972; Rabbit anti-GST, Santa Cruz, sc-459; Rabbit anti-phospho-PRAS40 T246, Invitrogen; Rabbit anti-phospho-PRAS40 S183, IBL; Mouse anti-HA, Santa Cruz, sc-7392; Rabbit anti-phospho-p70, Cell Signaling, 9205; Rabbit anti-p70, Cell Signaling, 2708; Mouse anti-p53, Cell Signaling, 2524; Mouse anti-p21, Cell Signaling, 2946; Rabbit anti-Bax, Cell Signaling 5023; Mouse anti-β-actin, Sigma, A2228.

Havel J.J., Li Z., Cheng D., Peng J, & Fu H. (2014). Nuclear PRAS40 couples the Akt/mTORC1 signaling axis to the RPL11-HDM2-p53 nucleolar stress response pathway. Oncogene, 34(12), 1487-1498.

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Immunofluorescence Staining Protocols for PRRSV Detection

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Mouse anti-PRRSV N protein and monoclonal J2 anti-dsRNA antibodies were purchased from Jeno Biotech Inc. (Chuncheon, South Korea) and Scicons (Hungary), respectively. Cellular DNA, mitochondria and lysosomes were stained with DAPI, MitoTracker Red and LysoTracker Red, respectively (all reagents were from Molecular Probes, OR, USA). Rabbit anti-calnexin, anti-LC3 and anti-IgG Alexa Fluor were purchased from Cell Signaling Technology. A goat anti-mouse IgG-Gold antibody was obtained from Sigma-Aldrich.

Zhang W., Chen K., Zhang X., Guo C., Chen Y, & Liu X. (2018). An integrated analysis of membrane remodeling during porcine reproductive and respiratory syndrome virus replication and assembly. PLoS ONE, 13(7), e0200919.

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Comprehensive Antibody Panel for Protein Analysis

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Havel J.J., Li Z., Cheng D., Peng J, & Fu H. (2014). Nuclear PRAS40 couples the Akt/mTORC1 signaling axis to the RPL11-HDM2-p53 nucleolar stress response pathway. Oncogene, 34(12), 1487-1498.

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Immunofluorescence Analysis of HA-Tagged Proteins

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At 24 h after transfection, cells were treated for 2 h with 50 μM MG-132 (in DMSO). Cell slides were fixed with 4% paraformaldehyde (in PBS) for 30 min at room temperature (RT), and permeabilized with 0.5% Triton X-100 (in PBS) for 20 min after washing with PBS. Slides were incubated with 5% bovine serum albumin at room temperature for 2 h and then with mouse anti-HA (1:100; Cell Signaling Technology) and rabbit anti-calnexin (1:100; Cell Signaling Technology) for 8 h at 4 °C, respectively. After three washes in PBS, donkey anti-mouse IgG-AlexaFlour 488 antibody (1:200; abs20014, Absin, Shanghai, China) and donkey anti-rabbit IgG-AlexaFlour 594 antibody (1:200; abs20021, Absin) was applied for 2 h. Nuclei were labeled with DAPI (in PBS) for 5 min. Cell slides were imaged using two-photon microscopy (Zeiss NLO 780, Carl Zeiss, Oberkochen, Germany).

Zhang L., Li Y., Qin L., Wu Y, & Lei B. (2021). Autosomal Recessive Retinitis Pigmentosa Associated with Three Novel REEP6 Variants in Chinese Population. Genes, 12(4), 537.

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Immunoblotting with Cell Signaling Antibodies

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Primary antibodies used in this study included rabbit anti-CD63 (System Biosciences, USA), rabbit anti-CD81 (System Biosciences, USA), rabbit anti-Calnexin (Cell Signaling, China), mouse anti-β-tubulin (Sigma), rabbit anti-phospho-Histone H3 (Ser10) (Milipore Sigma), rabbit anti-Phospho-Stat3 (Tyr705), rabbit anti-STAT3, rabbit anti-Cyclin D1, rabbit anti-Hes1 (Cell Signaling, China). Secondary antibodies used in this study were goat anti-rabbit or mouse HRP, donkey anti-rabbit or mouse Cy5, goat anti-rabbit or mouse Alexa 594, goat anti-human FITC or goat anti-rabbit or mouse Alexa 488 (Jackson Immuno Research, West grove, PA, USA).

Ren R., Sun H., Ma C., Liu J, & Wang H. (2019). Colon cancer cells secrete exosomes to promote self-proliferation by shortening mitosis duration and activation of STAT3 in a hypoxic environment. Cell & Bioscience, 9, 62.

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