SH-SY5Y cell line with deletion of calnexin was generated by CRISPR-Cas9–mediated genome editing according to the standard protocol described previously (Ran et al., 2013 (link)). We used four sgRNAs targeting four loci of calnexin to induce DNA double strand breaks. sgRNAs were individually cloned into the CRISPR vector. The sequences of sgRNAs were 5′-GCTTGGAACTGCTATTGTTG-3′, 5′-CCTCTTCAATGACATCGTCA-3′, 5′-TTTGACAGAGGAACTCTGTC-3′, and 5′-TATACTTCCCCTGTTGGAAC-3′. Calnexin−/− cells were verified by DNA sequencing (the PCR primers were forward, 5′-GTTTTAACGGGTACCCCCTGT-3′ and reverse, 5′-AGAGTGGGGGAAAGAA-3′), followed by Western blot analysis using Calnexin antibody (Abcam) to detect Calnexin proteins in the cells.
Calnexin antibody
Manufactured by Abcam
Sourced in United States
Calnexin antibody is a protein that binds to and identifies the calnexin protein, which is an endoplasmic reticulum chaperone involved in the folding and quality control of newly synthesized glycoproteins.
Automatically generated - may contain errors
Lab products found in correlation
Cd9 antibody, by Abcam (2 mentions)
Tsg101 antibody, by Abcam (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Laemilli buffer, by Bio-Rad (1 mentions)
Alexa fluor 568 conjugated goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Na 1.4 objective, by Zeiss (1 mentions)
12 bit orca er camera, by Hamamatsu Photonics (1 mentions)
Fitc conjugated goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Axiovert 200 microscope, by Zeiss (1 mentions)
Phosphatase inhibitor cocktail, by Ipsen (1 mentions)
Protease inhibitor cocktail, by Ipsen (1 mentions)
Ripa lysis buffer, by Ipsen (1 mentions)
Cd81 antibody, by Abcam (1 mentions)
Bca protein assay kit, by Takara Bio (1 mentions)
5 protocols using calnexin antibody
1
CRISPR-Cas9 Knockout of Calnexin in SH-SY5Y Cells
2
Exosomal miR-588 Characterization
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The morphology as well as the size distribution of Exos, Exos/miR-588, cRGD-Exos/miR-588 were characterized by transmission electron microscopy (TEM) and Nano-ZEN3600. The surface markers of exosomes were characterized using Western Blot. The antibodies were CD9 antibody (1:1000, Abcam), TSG-101 antibody (1:1000, Abcam), and Calnexin antibody (1:1000, Abcam). The miR-588 content of Exos and Exos/miR-588 were measured separately by qPCR to demonstrate that miR-588 had been successfully loaded.
3
Protein Quantification by Western Blot
4
Imaging of Transfected ATL2 Variants
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COS-7 cells were cultured on 12-mm glass coverslips in a 24-well plate in DMEM + 10% FBS + 1% penicillin-streptomycin. Transfections of HA-tagged WT or E555K, E556K, or R559E ATL2-1 in pGW1-CMV vector were performed using 1.5 µl Lipofectamine 2000 (Thermo Fisher Scientific) and the indicated amounts of DNA, following manufacturer’s instructions. 2 d after transfection, cells were fixed in 3% paraformaldehyde and costained with HA antibody Ab18181 (Abcam) at 1:500 and calnexin antibody (Abcam) at 1:200 using FBS as blocking agent. Secondary antibodies were Alexa Fluor 568–conjugated goat anti-mouse and FITC-conjugated goat anti-rabbit (Thermo Fisher Scientific). Images were obtained using a spinning-disk confocal scanhead (Yokagawa; PerkinElmer) mounted on an Axiovert 200 microscope (Zeiss) with a 100× 1.4-NA objective (Zeiss) and acquired using a 12-bit ORCA-ER camera (Hamamatsu Photonics). Maximal value projections of two to four sections at 0.2-µm spacing were acquired using Micromanager open-source software (University of California, San Francisco) and merged after adjustment using GIMP open-source software (University of California, Berkeley).
5
Extracellular Vesicle Protein Profiling
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The cartilage and chondrocytes were treated with the RIPA lysis buffer (Epizyme, PC101, China) added with a protease inhibitor cocktail (Epizyme, GRF101, China) and a phosphatase inhibitor cocktail (Epizyme, GRF102). The protein contents were calculated using a BCA protein assay kit (Takara, T9300A), and the samples was instantly boiled for 10 min with the addition of loading buffer. An equal quantity of protein extracts (20 μg) was placed onto a sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel for electrophoresis and transmitted to a PVDF membrane. Following that, the PVDF membrane was sequentially incubated with primary and secondary antibodies. Finally, the enhanced chemiluminescence (BI, 20-500-120) was used to react with secondary antibodies and the images were acquired. The mTOR-antibody (CST, 2983), P-p70S6 (CST, 9234), Calnexin-antibody (Abcam, ab133615, USA), CD9-antibody (Abcam, ab263019), CD81-antibody (Abcam, ab109201), TSG101-antibody (Abcam, ab125011), Alix-antibody (CST, 92880), Lamp2b-antibody (Abcam, ab18529), anti-rabbit IgG, HRP-linked Antibody (CST, 7074) were used as the antibodies.
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