Potassium hexacyanoferrate (III), methanol, sodium nitrite, and hydrochloric acid were purchased from Chempur (Poland). Phosphate-buffered saline (PBS), 4-aminobenzoic acid (4-ABA), N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), Tris-buffered saline (TBS), IPTG, HIS-Select® Nickel Affinity Gel, Tris-HCl, Glycerol, 4-aminothiphenol (4-ATP), glutaraldehyde (GA), bovine serum albumin (BSA), ethanol, demineralized/ultrapure water (ddH2O), hydrochloric acid (37%), sodium nitrite and ethylenediaminetetraacetic acid (EDTA) were purchased from Sigma Aldrich (Germany). Codon optimization for E. coli, ALICator Ligation Independent Cloning, B-PER Complete Bacterial Protein Extraction Reagent, Gene Art Gene Synthesis, Slide-A-Lyzer Dialysis Cassettes, Wedge Well Gel, Super Signal West Pico Plus Chemiluminescent Substrate, and 1-Step Turbo TMB-ELISA were purchased from Thermo Fisher Scientific (USA). pFastBac1 was purchased from Invitrogen (USA). 15 cm Chromatography column, Trans-Blot Turbo RTA Transfer Kit were purchased from Biorad (USA). Protein A-agarose affinity matrix was purchased from Roche (Swiss). Instant Blue was purchased from Expedeon (United Kingdom). Secondary goat anti-rabbit HRP antibodies were purchased from Jackson ImmunoResearch (United Kingdom). 96-well ELISA plate was purchased from Greiner Microlon (Germany).
Wedgewell gel
Manufactured by Thermo Fisher Scientific
Sourced in United States
The WedgeWell Gel is a laboratory equipment designed for electrophoresis applications. It provides a stable and consistent platform for the separation and analysis of biomolecules, such as proteins and nucleic acids.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (4 mentions)
Hydrochloric acid (hcl), by Chempur (1 mentions)
Sodium nitrite, by Chempur (1 mentions)
Slide a lyzer dialysis cassette, by Thermo Fisher Scientific (1 mentions)
Trans blot turbo rta transfer kit, by Bio-Rad (1 mentions)
Tris hcl, by Merck Group (1 mentions)
Pfastbac1, by Thermo Fisher Scientific (1 mentions)
Potassium hexacyanoferrate 3, by Chempur (1 mentions)
B per complete bacterial protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
1 step turbo tmb elisa, by Thermo Fisher Scientific (1 mentions)
His select nickel affinity gel, by Merck Group (1 mentions)
Instantblue, by Abcam (1 mentions)
Tris buffered saline (tbs), by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Glycerol, by Merck Group (1 mentions)
1 ethyl 3 3 dimethylaminopropyl carbodiimide, by Merck Group (1 mentions)
Peroxidase conjugated affinipure goat anti rabbit antibodies, by Jackson ImmunoResearch (1 mentions)
Ab92976, by Abcam (1 mentions)
Peroxidase conjugated mouse anti armenian hamster antibody, by Santa Cruz Biotechnology (1 mentions)
Anti β actin antibody, by Abcam (1 mentions)
4 protocols using wedgewell gel
1
Recombinant Protein Expression and Purification
2
NoV Capsid Protein Detection by Western Blot
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Samples containing NoV VLPs were loaded on 10–20% precast WedgeWell Gel (Thermo Scientific) and run at the constant voltage of 165 V. After electrophoresis, semi-dry electrotransfer of proteins onto polyvinylidene difluoride membranes was performed. Membranes were then blocked for 1 h in 5% semi-skimmed milk solution (5%milk/TBS/0.01% Tween20) and incubated overnight at 8 °C with rabbit anti-N terminal capsid protein of NoV antibodies (Abcam ab92976) (1:1000 in 5%milk/TBS/0.01%Tween20). The following day, the membranes were washed 3 times with washing buffer (TBS/0.01%Tween20) and incubated for 1 h at room temperature in a solution of Peroxidase-conjugated AffiniPure Goat Anti-Rabbit antibodies (Jackson Immuno Research) (1:4000 in 5%milk/TBS/0.01%Tween20). After washing (same as above) the reaction was developed with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Scientific).
3
Norovirus Capsid Protein and MUC1 Detection
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NoV VP1-MUC1, NoV VP1 and WT L. tarentolae cell lysates were loaded on 10–20% precast WedgeWell Gel (Thermo Fisher Scientific) and run at the constant voltage of 165 V. After electrophoresis, semi-dry electrotransfer of proteins onto polyvinylidene difluoride membranes was performed. Membranes were then blocked for 1 h in a 5% semi-skimmed milk solution (5%milk/TBS/0,01%Tween20) and incubated overnight with primary antibodies solution: Armenian hamster anti-MUC1 antibody (MA5-11,202, Thermo Fisher Scientific; 1:100 in 5%milk/TBS/0,01%Tween20) or rabbit anti-N terminal capsid protein of NoV antibodies (ab92976, Abcam; 1:1000 in 5%milk/TBS/0,01%Tween20). The following day, the membranes were washed (TBS/0,01%Tween20) and incubated with solution of Peroxidase-conjugated mouse anti-Armenian hamster antibody (Santa Cruz Biotechnology; 1:4000 in 5%milk/TBS/0,01%Tween20) or AffiniPure goat anti-rabbit antibodies (Jackson Immuno Research; 1:4000 in 5%milk/TBS/0,01%Tween20). A reaction was developed with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific).
4
Profiling Baculovirus-Infected Insect Cell Proteins
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Culture media from NoV-M2/NP baculovirus-infected Sf9 insect cells was harvested 96 h post-infection. Culture media from cells infected with an empty NoV platform was used as a control. Samples were loaded on 10%–20% precast WedgeWell Gel (Thermo Fisher Scientific, Waltham, USA) and run at the constant voltage of 165 V. After electrophoresis, semi-dry electrotransfer of proteins onto nitrocellulose membranes was performed using Trans-Blot Turbo RTA Mini 0.2 μm Nitrocellulose Transfer Kit (Bio-Rad, Hercules, USA). Membranes were then blocked for 1 h in a 5% semi-skimmed milk solution (5% milk/TBS/0.01% Tween 20) and incubated overnight with primary antibodies solution: rabbit polyclonal anti-NoV serum (obtained by vaccination of rabbit with insect cell-derived NoV VP1), mouse monoclonal anti-M2 (Santa Cruz Biotechnologies, Dallas, USA) rabbit anti-NP polyclonal antibody (Genetex, Irvine, USA), or anti-β-actin antibodies (Abcam, Waltham, USA) in 5% milk/TBS/0.01% Tween 20. Then, the membranes were washed (TBS/0.01% Tween 20) and incubated with a solution of AffiniPure goat anti-rabbit or anti-mouse antibodies (Jackson Immuno Research, Ely, United Kingdom) in 5% milk/TBS/0.01% Tween 20. The reaction was developed with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, USA).
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