Aneurysm samples were homogenized in protein extraction buffer (0.3 M sucrose; 25 mM Imidazole, 1 mM EDTA, pH 7.2 complete protease inhibitor cocktail 2 and 3, Sigma Aldrich, Søborg, Denmark). Samples were centrifuged for 10 min at 6000× g at 4 °C. Protein concentration was determined by Bicinchoninic Acid Kit for Protein Determination (Sigma Aldrich, Søborg, Denmark) using bovine serum albumin as the standard. A total of 12 µg protein samples and 1.25 µL recombinant MMP-2 (Sigma Aldrich, Søborg, Denmark) were mixed with an equal amount of 2× tris-glycine SDS sample buffer (Thermo Fischer) loaded onto a Novex zymogram gel containing 10% gelatin (Thermo Fisher, Slangerup, Denmark) and proteins were separated by gel electrophoresis at 125 V for 90 min. Proteins were then allowed to refold 30 min in renaturation buffer (Thermo Fisher, Slangerup, Denmark) followed by 24 h at 37 °C in developing buffer (Thermo Fischer, Slangerup, Denmark). Finally, undigested proteins in the gel were stained with simple blue stain (Thermo Fisher) for 30 min. White bands were inverted and quantified in Molecular Imager Image Lab (ChemiDoc WRS+, Biorad, Copenhagen, Denmark).
Simple blue stain
Manufactured by Thermo Fisher Scientific
Sourced in United States
Simple blue stain is a laboratory reagent used for staining and visualization of biological samples. It is a basic dye that binds to acidic components within cells, allowing for the detection and identification of cellular structures under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
Recombinant mmp 2, by Merck Group (2 mentions)
Opti mem 1, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Novex 4 12 tris glycinesds page gels, by Thermo Fisher Scientific (2 mentions)
Novex zymogram gel, by Thermo Fisher Scientific (1 mentions)
Tris glycine sds sample buffer, by Thermo Fisher Scientific (1 mentions)
Developing buffer, by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid kit for protein determination, by Merck Group (1 mentions)
Renaturation buffer, by Thermo Fisher Scientific (1 mentions)
Maxisorp 96 well plate, by Thermo Fisher Scientific (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Thermo Fisher Scientific (1 mentions)
Cacl2, by Thermo Fisher Scientific (1 mentions)
Imidazole, by Thermo Fisher Scientific (1 mentions)
Citric acid, by Thermo Fisher Scientific (1 mentions)
Freestyle 293 expression media, by Thermo Fisher Scientific (1 mentions)
O phenylenediamine dihydrochloride, by Merck Group (1 mentions)
Hrp linked peanut agglutinin, by Merck Group (1 mentions)
Valproic acid, by Merck Group (1 mentions)
5 protocols using simple blue stain
1
Gelatin Zymography for MMP-2 Activity
2
Influenza Virus Neuraminidase Activity Assay
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Rosetta 2 cells and SEC molecular weight standard were from Agilent. Imidazole, isopropyl β-D-1-thiogalactopyranoside (IPTG), 1 ml HisTrap Fast Flow crude columns, glutathione-sepharose, Tris, phosphate, citric acid, CaCl2, protease inhibitor cocktail (PIN), Sf-900 III serum free medium (Sf-900), FreeStyle 293 (293F) cells, FreeStyle 293 expression media, simple blue stain, Novex WedgeWell 4 to 12% Tris-Glycine SDS-PAGE gels, Novex sharp unstained protein standard, Maxisorp 96-well plates, and BCA protein assay kit were obtained from Thermo Fisher Scientific. Centrifugal filters (10 and 30 kDa), polyethylenimine, and 4-methylumbelliferyl-α-D-N-acetylneuraminide (MUNANA) were acquired from Amicon, Polysciences, and Cayman Chemical, respectively. Bovine fetuin, 4-methylumbelliferone sodium salt (4-MU), o-phenylenediamine dihydrochloride, valproic acid, and HRP-linked peanut agglutinin were purchased from Sigma. Low protein binding 96-well black clear bottom fluorescent assay plates were from Corning. Specific-pathogen-free hen’s eggs were purchased from Charles River Labs. The reassortant Influenza A viruses WSNH1N1/BR18 (N1 virus), which carries the HA and NA genes from the strain A/Brisbane/02/2018 (H1N1), and WSNN2-KS17 (N2 virus), which carries the N2 gene from the strain A/Kansas/14/2017, were generated for prior studies (53 , 72 ).
3
Quantitative Zymography for MMP2 Activity
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Proteins were extracted by homogenization in an extraction buffer. The bicinchoninic acid protein determination kit (Sigma-Aldrich, Søborg, Denmark) was used to determine protein concentrations using serum albumin as standard. AAA protein samples (12 µg) and 1.25 µL recombinant MMP2 (Sigma Aldrich, Søborg, Denmark) were loaded onto Novex zymogram gel, separated, renatured, and incubated for 24 h at 37 °C in a developing buffer, before undigested proteins were stained by a simple blue stain (Thermo Fischer, Waltham, MA, USA). White bands representing digested proteins were inverted and quantified in Molecular Imager Image Lab (ChemiDoc WRS+, Biorad, Copenhagen, Denmark) as described by Melin et al. [30 (link)].
4
Comprehensive Molecular Biology Protocol
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Dulbecco’s modified Eagle medium (DMEM),
fetal bovine serum (FBS),l -glutamine, penicillin/streptomycin
(P/S), Opti-MEM I (OMEM), Simple Blue Stain, Novex 4–12% Tris-glycine
SDS-PAGE gels, Novex Sharp Unstained Protein Standard, GeneRuler 1
kb Plus DNA Ladder, LB Medium Dehydrated Capsules, and the Phusion
High-Fidelity DNA Polymerase were all purchased from Thermo Fisher
Scientific. His-tagged Pfu X7 DNA Polymerase was prepared in-house
by immobilized metal affinity chromatography for routine PCR-based
bacterial colony screening. XL10-Gold Ultracompetent cells were acquired
from Agilent Technologies, Inc. Note XL10-Gold cells have a mutation
in the lacI gene promoter (lacIq), resulting in higher Lac repressor levels.57 (link) SIGMAFAST EDTA-free Protease Inhibitor cocktail
tablets and IPTG were obtained from Sigma-Aldrich. DpnI, TransIT-LT1 transfection reagent, and 2′-(4-methylumbelliferyl)-α-d -N-acetylneuraminic acid (MUNANA) were obtained
from New England Biolabs, Mirus Bio, and Cayman Chemicals, respectively.
Specific-pathogen-free (SPF) eggs and turkey red blood cells (TRBCs)
were purchased from Charles River Labs and the Poultry Diagnostic
and Research Center (Athens, GA), respectively. All cloning and screening
primers (Table S1 ) used in this study were
synthesized by Integrated DNA Technologies.
fetal bovine serum (FBS),
(P/S), Opti-MEM I (OMEM), Simple Blue Stain, Novex 4–12% Tris-glycine
SDS-PAGE gels, Novex Sharp Unstained Protein Standard, GeneRuler 1
kb Plus DNA Ladder, LB Medium Dehydrated Capsules, and the Phusion
High-Fidelity DNA Polymerase were all purchased from Thermo Fisher
Scientific. His-tagged Pfu X7 DNA Polymerase was prepared in-house
by immobilized metal affinity chromatography for routine PCR-based
bacterial colony screening. XL10-Gold Ultracompetent cells were acquired
from Agilent Technologies, Inc. Note XL10-Gold cells have a mutation
in the lacI gene promoter (lacIq), resulting in higher Lac repressor levels.57 (link) SIGMAFAST EDTA-free Protease Inhibitor cocktail
tablets and IPTG were obtained from Sigma-Aldrich. DpnI, TransIT-LT1 transfection reagent, and 2′-(4-methylumbelliferyl)-α-
from New England Biolabs, Mirus Bio, and Cayman Chemicals, respectively.
Specific-pathogen-free (SPF) eggs and turkey red blood cells (TRBCs)
were purchased from Charles River Labs and the Poultry Diagnostic
and Research Center (Athens, GA), respectively. All cloning and screening
primers (
synthesized by Integrated DNA Technologies.
5
Influenza Virus Hemagglutinin and Neuraminidase Assay
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Dulbecco’s Modified Eagles Medium (DMEM), fetal bovine serum (FBS), L-glutamine, penicillin/streptomycin (P/S), Opti-MEM I (OMEM), anti-goat IgG HRP-linked secondary antibody, Simple Blue Stain, Novex 4–12% Tris-Glycine SDS-PAGE gels, dithiothreitol (DTT) Immulon-2HB 96-well plates, Nickel coated 96-well plates and Lipofectamine 2000 transfection reagent were all purchased from Thermo Fisher Scientific. Zanamivir and 2’-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid (MUNANA) were acquired from Moravek Inc and Cayman Chemicals, respectively. β-propiolactone, formaldehyde, o-Phenylenediamine dihydrochloride (OPD), and anti-mouse IgG HRP linked secondary were purchased from Sigma. Anti-rabbit IgG HRP-linked secondary antibody and 0.45-μm polyvinylidene difluoride (PVDF) membrane were obtained from GE healthcare. Specific-Pathogen-Free (SPF) eggs and turkey red blood cells (TRBCs) were purchased from Charles River Labs and the Poultry Diagnostic and Research Center (Athens, GA), respectively. The H1N1 A/Brisbane/02/2018 field isolate (WT) and CVV (IVR-190) were kindly provided by the WHO. Rabbit Antisera against NA was generated by Agrisera (Sweden) using NA-WSN residues 35–453 isolated from E. coli inclusion bodies [29 (link)]. Polyclonal goat antiserum against the H1N1 influenza virus A/Fort Monmouth/1/1947 (NR-3117) was obtained from BEI Resources, NIAID, NIH.
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