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Anti calnexin antibody

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Anti-calnexin antibody is a protein that binds to the calnexin protein, which is a chaperone protein involved in the folding and quality control of newly synthesized glycoproteins in the endoplasmic reticulum.

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Lab products found in correlation

Anti gm130, by BD (2 mentions) Anti alix antibody, by Abcam (2 mentions) Anti cd63 antibody, by Abcam (2 mentions) Ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions) Anti gapdh antibody, by Cell Signaling Technology (1 mentions) Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions) Eclipse ti confocal microscope, by Nikon (1 mentions) Bodipy 493 503, by Thermo Fisher Scientific (1 mentions) Chambered glass slides, by BD (1 mentions) Proteinase phosphatase inhibitors, by Thermo Fisher Scientific (1 mentions) Ripa lysis buffer, by Thermo Fisher Scientific (1 mentions) Anti flag tag m2 antibody, by Merck Group (1 mentions) Anti actin antibody, by Merck Group (1 mentions) Nitrocellulose membrane, by Cytiva (1 mentions) Ecl kit, by Cytiva (1 mentions) Anti ha tag antibody, by Fortrea (1 mentions) Mab4a, by Synaptic Systems (1 mentions) Goat anti rabbit cy3, by Dianova (1 mentions) Hybond c extra membrane, by Cytiva (1 mentions) Anti rabbit secondary antibody, by Agilent Technologies (1 mentions)

Spelling variants of this product

Calnexin (140 citations) Anti-Calnexin (92 citations) Calnexin antibody (5 citations) Rabbit anti-calnexin antibody (5 citations) Mouse anti-Calnexin antibody (2 citations) Rabbit anti-calnexin polyclonal antibody (2 citations)

16 protocols using anti calnexin antibody

1

Western Blot Analysis of DNA Damage Response Proteins

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Whole cell lysates were prepared by boiling cell pellets for 10 min in a SDS lysis buffer (2% SDS, 10% glycerol, 10 mM DTT, 62 mM Tris–HCl pH 6.8, 10 μg/ml pepstatin and 1 μg/ml leupeptin). Proteins were quantitated, and 50 μg of protein were separated by SDS-PAGE gels, transferred to the nitrocellulose membrane, blocked with 5% FBS at room temperature for 1 h on a shaker. The membranes were incubated with the anti-Pol η antibody (1: 1000, A301–231A, Bethyl Laboratories), anti-REV1 antibody (1: 1000, PA5–46793, Thermo Fisher Scientific), anti-Ubiquityl-PCNA antibody (1:1000, #13439, Cell Signaling Technology), anti-Calnexin antibody (1:2000, ab92573, abcam), or anti-GAPDH antibody (1:2000, #97166, Cell Signaling Technology) overnight at 4°C. The membranes were washed with TBST, then incubated in an HRP-conjugated secondary antibody for 1 h at room temperature. After wash, the proteins were visualized by ECL Western blotting substrate (Thermo Fisher Scientific).
Wu D., Banerjee A., Cai S., Li N., Han C., Bai X., Zhang J, & Wang Q.E. (2021). Determination of DNA lesion bypass using a ChIP-based assay. DNA repair, 108, 103230.
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2

Visualizing Cellular Organelles by Immunofluorescence

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Cells were plated on chambered glass slides (Falcon, Corning) and incubated in 500 μl growth medium per chamber. Immediately prior to staining, the media were removed from each chamber, cells were washed with PBS (pH 7.4), and then fixed with 3% (v/v) formaldehyde. For performing immunofluorescence, the fixed cells were washed with PBS, permeabilized with 0.3% (v/v) Triton-X, and then incubated with 5% goat serum for 1 h at room temperature. Subsequently anti-calnexin antibody (1:250, Abcam) was added to the cells. After 2 h incubation, cells were rinsed with PBS, and treated with goat anti-rabbit IgG, Alexa Fluor 633 (1:500, Life Technologies) for 1 h. For staining the lipid droplets, cells were rinsed again with PBS and incubated with 10 μg/ml BODIPY® 493/503 in dark. The stained cells were then washed with PBS, and the chamber was slowly removed before mounting the cover slips using ProLong® Gold antifade reagent with DAPI (ThermoFisher Scientific). The slides were then imaged under Nikon Eclipse Ti confocal microscope with 400× magnification.
Anaganti N., Chattopadhyay A., Poirier J.T, & Hussain M.M. (2022). Generation of hepatoma cell lines deficient in microsomal triglyceride transfer protein. Journal of Lipid Research, 63(9), 100257.
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3

Western Blot Protein Detection Protocol

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Nuclei from mouse liver tissues were prepared as previously described (Jeon et al., 2013 (link); Tarling et al., 2015 (link)). Total cellular and tissue proteins were extracted with RIPA lysis buffer (Thermo Scientific) with proteinase/phosphatase inhibitors (Thermo Scientific). Lysate was separated by 4–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins on the gel were electrophoretically transferred onto nitrocellulose membranes (Amersham Biosciences). The membranes were blocked in PBS supplemented with 5% non-fat milk to quench nonspecific protein binding, and then incubated with the indicated antibodies. Horse radish peroxidase-conjugated anti-mouse and anti-rabbit IgG (Jackson) were used as secondary antibodies. The immune signal was visualized using the ECL kit (Amersham Biosciences).
The following primary antibodies were used: anti-FLAG tag M2 antibody (Sigma, F3165), anti-HA tag antibody (Covance, MMS-101R), anti-V5 tag antibody (Life Technologies, R960-25), anti-actin antibody (Sigma, A2066), anti-HSV tag antibody (Millipore, 69171), anti-calnexin antibody (Abcam, ab10286), anti-SREBP-2 antibody (Millipore, MABS1988).
Zhang L., Rajbhandari P., Priest C., Sandhu J., Wu X., Temel R., Castrillo A., de Aguiar Vallim T.Q., Sallam T, & Tontonoz P. (2017). Inhibition of cholesterol biosynthesis through RNF145-dependent ubiquitination of SCAP. eLife, 6, e28766.
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4

Subcellular Localization of GlyR Variants

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COS7 cells transiently expressing GlyR α1 (wt) or GlyR α1 variants were stained in permeabilized cells with primary antibodies MAb4a (Synaptic Systems, Göttingen Germany) and a polyclonal anti-calnexin antibody (1:500, Abcam, Cambridge, UK) for ER staining. The detection of ERGIC was done by the monoclonal ERGIC53 antibody (1:500, Enzo Life Science, Lörrach, Germany) and cis-Golgi stainings using a monoclonal antibody anti-GM130 (1:500, BD Transduction Laboratories, Heidelberg, Germany) together with a GlyR α1 specific antibody (Chemicon, Darmstadt, Germany). Secondary antibodies used were goat anti-mouse Cy3/Alexa488 and, goat anti–rabbit Cy3 (Dianova, Hamburg, Germany) diluted 1:500. All stainings were subjected to confocal microscopy on a DMIRE2 confocal microscope.
Atak S., Langlhofer G., Schaefer N., Kessler D., Meiselbach H., Delto C., Schindelin H, & Villmann C. (2015). Disturbances of Ligand Potency and Enhanced Degradation of the Human Glycine Receptor at Affected Positions G160 and T162 Originally Identified in Patients Suffering from Hyperekplexia. Frontiers in Molecular Neuroscience, 8, 79.
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5

Western Blot Analysis of siRNA Samples

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siRNA assays samples were electroblotted onto a Hybond-C Extra membrane (Amersham, Chalfont St Giles, UK), and antibody recognition was performed using in-house or commercial antibodies. A blocking solution was added to cover the membrane: the buffer contained 3–5% lyophilised milk for alimentary use, 1X PBS and 0.1% Tween20. The membrane was incubated for at least one hour and washed again with 1X PBS. The membrane was consequently incubated with the specific primary antibody, diluted in 1X PBS + 0.1% Tween20 and 3% BSA, for 1–2 hours. The membrane was washed with 1X PBS three times, and incubated with the appropriate secondary antibodies for 1–2 hours and washed again. Protein bands were detected using an enhanced chemiluminescence kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. The following antibodies were used in this study: Polyclonal Anti-PTB antibody produced in rabbit (in house from ICGEB, Trieste, Italy); dilution: 1:1000. Polyclonal Anti-hnRNP H antibody produced in rabbit (SIGMA; catalogue number: SAB4501422); dilution: 1:500 Anti-Calnexin antibody (Abcam; catalogue number: ab22595); dilution: 1:1000. Anti-Rabbit secondary antibody (Dako); dilution: 1:2000.
Mosca S., Raponi M., Meneghello A., Buratti E., Woods C.G, & Baralle D. (2017). Human NDE1 splicing and mammalian brain development. Scientific Reports, 7, 43504.
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6

Established Cell Lines and Antibodies

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The human TNBC cell line (MDA-MB-231) and murine macrophage (Raw264.7) were obtained from the Korean Cell Line Bank (Seoul, Korea). All cells were grown in Roswell Park Memorial Institute (RPMI) 1640 medium (WelGENE, Daegu, Korea) containing 10% fetal bovine serum (FBS) and supplemented with a 1% antibiotic solution containing penicillin and streptomycin (Thermo Fisher Scientific, Waltham, MA, USA). Cells were cultured in a 5% CO2 incubator at 37°C. The primary antibodies used in this study were anti-cytokeratin 8/18/19 antibody, anti-GFP antibody, anti-RFP antibody, anti-CD63 antibody, anti-ALIX antibody, and anti-calnexin antibody, purchased from Abcam (Cambridge, MA, USA). Anti-NOS2 antibody, anti-CD206 antibody, anti-arginase-1 antibody, and anti-GAPDH antibody were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). Anti-β-actin antibody was purchased from Sigma (St. Louis, MO, USA).
Piao Y.J., Kim H.S., Hwang E.H., Woo J., Zhang M, & Moon W.K. (2017). Breast cancer cell-derived exosomes and macrophage polarization are associated with lymph node metastasis. Oncotarget, 9(7), 7398-7410.
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7

Biotinylation and IP of IP3R3 and Calnexin

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A total of 1 × 107 D1 cells were biotinylated with 1 mg of biotin (EZ-Link sulfo-NHS-SS-biotin, Thermo Fisher Scientific) for 30 min at 37°C. Cells were then lysed with a buffer containing 50 mM tris-HCl (pH 7.4), 150 mM NaCl, 10% glycerol, and 1% NP-40 supplemented with protease and phosphatase inhibitor cocktails (Roche). Cell debris were removed by centrifugation at 16,000g for 15 min (4°C). The immunoprecipitations were performed using Dynabeads Protein G Immunoprecipitation Kit according to the manufacturer’s instructions. For IP3R3 immunoprecipitation, 10 μg of anti-IP3R3 (BD Biosciences, mouse clone 2, catalog no. 610312) or 10 μg of anti-IP3R3 (Millipore, rabbit polyclonal, catalog no. ab9076) was used. For calnexin immunoprecipitation, 10 μg of anti-calnexin antibody (Abcam, rabbit polyclonal, catalog no. ab2259) was used. The samples were run on a 10% polyacrylamide gel, and SDS-polyacrylamide gel electrophoresis was performed following standard procedures. After protein transfer, nitrocellulose membrane was incubated with streptavidin to reveal biotin-IP3R3 and with anti-calnexin antibody and developed using an ECL substrate reagent (Thermo Fisher Scientific).
Marongiu L., Mingozzi F., Cigni C., Marzi R., Di Gioia M., Garrè M., Parazzoli D., Sironi L., Collini M., Sakaguchi R., Morii T., Crosti M., Moro M., Schurmans S., Catelani T., Rotem R., Colombo M., Shears S., Prosperi D., Zanoni I, & Granucci F. (2021). Inositol 1,4,5-trisphosphate 3-kinase B promotes Ca2+ mobilization and the inflammatory activity of dendritic cells. Science signaling, 14(676), eaaz2120.
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8

Immunohistochemical Analysis of Neural Markers

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Immunohistochemistry was carried out as previously described (Goncalves et al., 2005 (link)).
Antibodies used were: mouse monoclonal anti-βIII tubulin (Promega, 1:1000); chicken polyclonal anti-GFAP (Abcam, 1:300); rabbit polyclonal anti-GFAP (DAKO, 1:2500); mouse monoclonal anti-GFAP (Sigma, 1:100); rabbit polyclonal anti-RARβ (Santa Cruz Biotechnology, Inc., 1:100); Rabbit polyclonal anti-NG2 (Millipore, 1:100); goat polyclonal anti-aldehyde dehydrogenase1A2 (Raldh2) (Santa Cruz Biotechnology, Inc., 1:100); goat polyclonal anti-alcohol dehydrogenase 7 (class IV) (ADH7) (Santa Cruz Biotechnology, Inc., 1:50); chicken polyclonal anti-microtubule associated protein 2 (MAP2) (Abcam, 1:1000); mouse monoclonal AC15 anti-beta actin (1:10,000, Sigma-Aldrich), rabbit polyclonal anti-AIP1/Alix (1:1000 for western blotting and 1:100 for immunocytochemistry, Millipore); anti-Calnexin antibody (1:1000 Abcam); chicken polyclonal anti-GFP antibody (Abcam, 1:500). Secondary antibodies for immunohistochemistry were AlexaFluor™ 594, AlexaFluor™ 488 and AlexaFluor™ 647 (1:1000, Molecular Probes, Life Technologies). DAPI was used to stain nuclei (1 μg/mL, Sigma Aldrich).
Goncalves M.B., Wu Y., Trigo D., Clarke E., Malmqvist T., Grist J., Hobbs C., Carlstedt T.P, & Corcoran J.P. (2018). Retinoic acid synthesis by NG2 expressing cells promotes a permissive environment for axonal outgrowth. Neurobiology of Disease, 111, 70-79.
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9

Immunoprecipitation of GFP and Calnexin

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Whole cell extracts were prepared as described above. Total protein concentration was measured with Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). 4 mg of whole cell extract was used per immunoprecipitation (IP). IP with GFP nanobody sepharose beads (Chromotek) was performed as described above. For Calnexin-IP, 10 μg of anti-Calnexin antibody from Abcam per IP was used. Input and IP samples were separated by SDS–PAGE and transferred to nitrocellulose membrane, followed by immunostaining. The antibodies used for immunostaining: anti-GFP (1:50; HMGU monoclonal antibody facility) or anti-Calnexin (1:1000; ab22595, Abcam) primary antibodies followed by horseradish peroxidase (HRP)-conjugated secondary antibodies goat anti-mouse (1:1500, P0447, Dako) and anti-rabbit (1:100000, 111-035-003, Jackson Laboratory) correspondingly.
Ignatova V.V., Jansen P.W., Baltissen M.P., Vermeulen M, & Schneider R. (2019). The interactome of a family of potential methyltransferases in HeLa cells. Scientific Reports, 9, 6584.
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10

Antibody Staining and Organelle Labeling

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Antibodies against p65 (C-20), SNF2H (H300), Tubulin (TU-02) and HA-probe (HA.C5) were acquired from Santa Cruz Biotechnology. Anti-JMJD8 and PDIA3 antibodies were purchased from Abnova. The anti-calnexin antibody was purchased from Abcam. Antibodies against EEA1 (C45B10), RCAS1 (D2B6N), AIF (D39D2), Kinectin 1 (D5F7J) and Lyric/Metadherin (MTDH) (2F11C3) were purchased from Cell Signaling Technology. LysoTracker® Red DND-99 and ER-Tracker™ Red dye were purchased from Invitrogen.
Yeo K.S., Tan M.C., Lim Y.Y, & Ea C.K. (2017). JMJD8 is a novel endoplasmic reticulum protein with a JmjC domain. Scientific Reports, 7, 15407.
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