Anti-Pan-Actin (#4968), Anti-ERK1/2 (#9102), anti-pERK1/2 Thr 202/Tyr204 (#9101), anti-AKT (#4685), anti-pAKT Ser473 (#9271 all from Cell Signaling), anti-FATP3 (Proteintech 12943-1-AP), anti-FATP4 (Abnova H00010999-M01), anti-PKC-θ (BD Transduction lab 610089), anti-Na,K ATPase A1 (Novus Biologicals NB300-146SS) and anti-HIBADH (Proteintech 13466-1-AP) antibodies were used for Western blot (1:1000 dilution). Anti-Occludin-1 (Abcam ab31721) and anti-Calnexin (Thermo MA3-27) antibodies were used for immunostaining (1:200 dilution). Mitotracker® Red CMXRos (Cell Signaling 9082S) was used for mitochondria staining. Sulfo-N-succinimidyl Oleate (sc-208408) was purchased from Santa Cruz. Akt VIII (#124018) was purchased from Calbiochem. CHC (#5029) was purchased from Tocris. Recombinant VEGFA (#293-VE-010) and VEGFB (#767-VE-010) were purchased from R&D systems. Human insulin (Humulin R U-100) was purchased from Harvard Drug Group (#821501). Membrane filters (MWCO 3 kDa #Z677094), Calcimycin (#C9275), 2,4-Dinitrophenol (#D198501), 2-deoxyglucose (#D8375), activated charcoal (#C4386) and other chemicals were purchased from Sigma unless otherwise stated.
Anti calnexin
Manufactured by Thermo Fisher Scientific
Sourced in United States
Calnexin is a molecular chaperone protein that is involved in the quality control of protein folding in the endoplasmic reticulum (ER). Anti-Calnexin is an antibody that specifically binds to and detects the presence of Calnexin in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd63, by Thermo Fisher Scientific (5 mentions)
Anti rab27a, by Thermo Fisher Scientific (3 mentions)
V3 western workflow, by Bio-Rad (3 mentions)
Ripa lysis and extraction buffer, by Thermo Fisher Scientific (3 mentions)
Precast polyacrylamide gel, by Bio-Rad (3 mentions)
Activated charcoal, by Merck Group (2 mentions)
2 deoxyglucose, by Merck Group (2 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti pan actin, by Cell Signaling Technology (2 mentions)
Anti fatp3, by Proteintech (2 mentions)
Anti fatp4, by Abnova (2 mentions)
Sulfo n succinimidyl oleate, by Santa Cruz Biotechnology (2 mentions)
Anti erk1 2, by Cell Signaling Technology (2 mentions)
Calcimycin, by Merck Group (2 mentions)
2 4 dinitrophenol, by Merck Group (2 mentions)
Anti p erk1 2 thr202 tyr204, by Cell Signaling Technology (2 mentions)
Anti pkc θ, by BD (2 mentions)
Anti hibadh, by Proteintech (2 mentions)
Anti occludin 1, by Abcam (2 mentions)
Recombinant vegfa, by R&D Systems (2 mentions)
14 protocols using anti calnexin
1
Western Blot and Immunostaining Techniques
2
Optimized Exosome Characterization Protocol
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Gold (III) chloride hydrate (HAuCl4—Cat No. 254169), trisodium citrate dihydrate (Cat No. S1804), poly(ethylene glycol) 2-mercaptoethanol ether acetic acid (PEG 3500 Da—Cat No. 757837), 2-(N-morpholino) ethanesulfonic acid (MES—Cat No. M5287), N-hydroxysuccinimide (NHS—Cat No. 56480), N-(3-dimethylaminopropyl)-N-ethyl carbodiimide (EDC—Cat No. 39391), and bovine serum albumin (Cat No. 05470) were procured from Sigma-Aldrich (St. Louis, MO, USA). Nonfat milk powder and clot activator tubes (4 mL) were procured from local vendors. Amersham ECL reagent (Cat No. RPN2235) was obtained from GE HealthCare (Chicago, IL, USA). All other reagents and consumables were of reagent grade. Primary antibodies anti-HSP70 (Cat No. MAb 33-3800), anti-CD63 (Cat No. BS-1523R) and anti-calnexin (Cat No. MA5-15389) were procured from Thermo Fisher (Waltham, MA, USA). Secondary antibodies anti-mouse (Cat No. ab131368) HRP and anti-rabbit (Cat No. ab131366) HRP were obtained from Abcam (Cambridge, UK).
3
Exosome Protein Characterization from hASCs and PRP
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The extracted proteins in the exosomes derived from
hASCs and PRP were isolated using a 10% sodium
dodecyl sulfate PAGE (SDS-PAGE) gel, and then
transferred to a nitrocellulose membrane. The blots were
blocked with 5% skimmed milk, then washed three times
with a TBST buffer, and incubated at 4ºC for 15 hours
with primary antibodies such as (anti-CD81, 1:100;
Thermo Fisher Scientific, Waltham, MA, USA), (anti-CD9, 1:100; Thermo Fisher Scientific, Waltham, MA,
USA), (monoclonal anti-TSG101, 1:100; Thermo Fisher
Scientific, Waltham, MA, USA) , (anti-CD63, 1:100;
Thermo Fisher Scientific, Waltham, MA, USA) and (anti-calnexin, 1:100; Thermo Fisher Scientific, Waltham, MA,
USA). In the next step, the blots were detected by their
corresponding secondary antibodies. Biorad’s ChemiDoc
MP Imaging System was used for band detection.
hASCs and PRP were isolated using a 10% sodium
dodecyl sulfate PAGE (SDS-PAGE) gel, and then
transferred to a nitrocellulose membrane. The blots were
blocked with 5% skimmed milk, then washed three times
with a TBST buffer, and incubated at 4ºC for 15 hours
with primary antibodies such as (anti-CD81, 1:100;
Thermo Fisher Scientific, Waltham, MA, USA), (anti-CD9, 1:100; Thermo Fisher Scientific, Waltham, MA,
USA), (monoclonal anti-TSG101, 1:100; Thermo Fisher
Scientific, Waltham, MA, USA) , (anti-CD63, 1:100;
Thermo Fisher Scientific, Waltham, MA, USA) and (anti-calnexin, 1:100; Thermo Fisher Scientific, Waltham, MA,
USA). In the next step, the blots were detected by their
corresponding secondary antibodies. Biorad’s ChemiDoc
MP Imaging System was used for band detection.
4
Neuroblastoma Cell Culture Protocols
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miR-2110 mimic, siRNAs and negative control oligos were purchased from Dharmacon. Rabbit anti-βIII-tubulin, anti-GAP43, anti-NSE, anti-TSKU, anti-calnexin, and HRP-conjugated anti-rabbit IgG antibodies were obtained from Thermo Fisher Scientific. Anti-cleaved PARP was purchased from Cell Signaling Technology. BE(2)-C, SKNDZ, CHLA-90, and SKNFI cells were from the American Type Culture Collection (ATCC). Kelly cells were obtained from the cell line repository at the Greehey Children’s Cancer Research Institute at the University of Texas Health San Antonio. Cells were grown in DMEM/F12 (Corning Cellgro) supplemented with 10% Equafetal bovine serum (Atlas Biologicals).
5
Western Blot and Immunostaining Techniques
6
Protein Expression Analysis Protocol
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Antibodies used were: anti-β-actin (#PA1-16889), anti-p16 (#PA5-16639), anti-p21 (#PA5-701151), anti-p27 (#PA5-13254), anti-p53 (#700439), anti-phospho-NF-κB (#PA5-37658), anti-NuMA (#PA132451), anti-calnexin (#MA3-027) (Thermo Fisher Scientific), anti-Bcl-2 (#sc-7382) (Santa Cruz), anti-active caspase 3 (#NB100-56113) (Novus Biologicals), anti-γH2AX (#CS208203) (Merck Millipore). Secondary HRP-conjugated: anti-mouse (#A9044) and anti-rabbit (#A0545) (Sigma).
7
Western Blot Analysis of Extracellular Vesicles
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Cell lines and EVs were lysed in RIPA buffer (150 mM NaCl, 0.5% sodium deoxycholate (Sigma-Aldrich–Merck, St. Louis, MO, USA), 1% Triton X-100 (Sigma-Aldrich–Merck), 50 mM Tris, pH 8.0, 0.1% sodium dodecyl sulphate, cocktails of protease inhibitors (cOmplete ULTRA Tablets, Mini, EDTA-free, EASYpack, Sigma-Aldrich–Merck)).
The following antibodies were used: anti-Alix (1A12, sc-53540, Santa Cruz Biotechnology; diluted 1:1000), anti-calnexin (MA3-027, Thermo Fisher Scientific; diluted 1:500), anti-beta-actin (AC-74, Sigma-Aldrich–Merck; diluted 1:5000), anti-Hsp70 (cmHsp70.1, produced in laboratory by Prof. Gabriele Multhoff [33 (link)]; diluted 1:1000), anti-Hsp60 (mouse monoclonal antibody ab13532, Abcam, Cambridge, UK), and anti-Rab5 (R4654, Sigma-Aldrich–Merck; diluted 1:500), all diluted in 5% milk in T-TBS. HRP-conjugated polyclonal rabbit anti-mouse immunoglobulin (P026002-2, Agilent–DAKO, Santa Clara, CA, USA) and HRP-conjugated polyclonal swine anti-rabbit immunoglobulin (P021702-2, Agilent–DAKO) diluted 1:2000 in 5% milk in T-TBS were used as secondary antibodies. Immunoreactive protein signals were detected using a Pierce ECL Western Blotting Substrate (Cat. no. 32106, Thermo Fisher Scientific) and captured using a ChemiDoc Imaging System (Bio-Rad, Hercules, CA, USA).
The following antibodies were used: anti-Alix (1A12, sc-53540, Santa Cruz Biotechnology; diluted 1:1000), anti-calnexin (MA3-027, Thermo Fisher Scientific; diluted 1:500), anti-beta-actin (AC-74, Sigma-Aldrich–Merck; diluted 1:5000), anti-Hsp70 (cmHsp70.1, produced in laboratory by Prof. Gabriele Multhoff [33 (link)]; diluted 1:1000), anti-Hsp60 (mouse monoclonal antibody ab13532, Abcam, Cambridge, UK), and anti-Rab5 (R4654, Sigma-Aldrich–Merck; diluted 1:500), all diluted in 5% milk in T-TBS. HRP-conjugated polyclonal rabbit anti-mouse immunoglobulin (P026002-2, Agilent–DAKO, Santa Clara, CA, USA) and HRP-conjugated polyclonal swine anti-rabbit immunoglobulin (P021702-2, Agilent–DAKO) diluted 1:2000 in 5% milk in T-TBS were used as secondary antibodies. Immunoreactive protein signals were detected using a Pierce ECL Western Blotting Substrate (Cat. no. 32106, Thermo Fisher Scientific) and captured using a ChemiDoc Imaging System (Bio-Rad, Hercules, CA, USA).
8
EV and HCMV Protein Characterization
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Total proteins were extracted from both EV and HCMV fractions, according to RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific, Waltham, MA). 10 μg of proteins were loaded on a 4–20% precast polyacrylamide gel (Bio-Rad Laboratories, Hercules, CA) and separated by SDS-PAGE, then transferred to PVDF membranes and probed with anti-CD63 (1 μg/ml, Thermo Fisher Scientific, Waltham, MA), anti-Calnexin (1 μg/ml, Thermo Fisher Scientific, Waltham, MA), anti-Rab27A (1 μg/ml, Thermo Fisher Scientific, Waltham, MA), anti-HCMV capsid MCP (2 μg/ml), and anti-HCMV tegument pp150 (2 μg/ml) monoclonal primary anti-mouse monoclonal antibodies (clones 28–4 and 36–14) and then goat peroxidase-conjugated anti-mouse IgG secondary anti-body (Bio-Rad Laboratories, Hercules, CA). Peroxidase activity and digital images were detected by using V3 Western Workflow™ (Bio-Rad Laboratories, Hercules, CA).
9
Extracellular Vesicle Protein Profile Analysis
10
EV and HCMV Protein Characterization
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