The substrate was whole unpasteurized cow’s milk. It was diluted in a ratio of 1:4 with a 20 mM Na-acetate buffer (pH = 5.65) containing 0.01% NaN3. The substrate was used on the day of preparation. A total of 250 μL of the substrate was mixed with 5 μL of rChn with an activity of ≈1000 AU/mL. The resulting enzyme–substrate mixtures were thoroughly stirred and incubated at 35 °C for 1 h. After the incubation, the mixtures were blended with a sample buffer for SDS-PAGE (“Serva”, Heidelberg, Germany) in the ratio of 1:1 and heated in a boiling-water bath for 90 s. Then, the samples were studied using electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) [41 (link)]. The LMW-SDS Marker Kit (“GE Healthcare”, USA) was used as molecular mass (MM) markers. In the control samples, instead of a rChn solution, 5 μL of a 20 mM Na-acetate buffer (pH 5.65) containing 0.01% NaN3 was added. The control samples were incubated for 1 h either at room temperature (control 1) or at 35 °C (control 2).
Lmw sds marker kit
Manufactured by GE Healthcare
Sourced in United States
The LMW-SDS Marker Kit is a set of pre-stained molecular weight standards for use in SDS-PAGE analysis. The kit provides a range of low molecular weight proteins that can be used to determine the molecular weights of unknown protein samples.
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Lab products found in correlation
Sds page, by Serva Electrophoresis (1 mentions)
Coomassie brilliant blue g 250, by Merck Group (1 mentions)
Hybond c extra, by GE Healthcare (1 mentions)
Bcip nbt, by Merck Group (1 mentions)
Mini protean tetra, by Bio-Rad (1 mentions)
Coomassie brilliant blue, by GE Healthcare (1 mentions)
Protease inhibitor cocktail tablets edta free, by Merck Group (1 mentions)
Sephacryl s 300 hr, by Merck Group (1 mentions)
Imagescaner 3, by GE Healthcare (1 mentions)
Umax powerlook 1120 scanner, by GE Healthcare (1 mentions)
Trypsin, by Promega (1 mentions)
Glass bead, by Merck Group (1 mentions)
Tween 80, by BD (1 mentions)
Complete edta free protease inhibitor cocktail, by Roche (1 mentions)
Amersham hybond ecl nitrocellulose membrane, by GE Healthcare (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Acquity uplc system, by Waters Corporation (1 mentions)
8 protocols using lmw sds marker kit
1
Characterization of Recombinant Chymosin Activity
2
SDS-PAGE and Immunoblotting for Fungal Proteins
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The purified rPbHsp60 and rgp43 were applied to a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a Mini Protean Tetra (Bio-Rad, USA). The gel protein bands were stained with Coomassie Brilliant Blue G-250 (Sigma-Aldrich, USA) or transferred to nitrocellulose membranes (Hybond-C Extra, GE Healthcare, USA). Proteins with known molecular weights were used as standards (LMW-SDS Marker Kit; GE Healthcare). Membranes containing 1 µg of rPbHsp60 or 1 µg of rgp43 were blocked with 3% gelatin in Tris-buffered saline (20 mM Tris-HCl, 150 mM NaCl, pH 7.2) containing 0.05% of Tween-20 (TBS-T), overnight at 4°C. Then, these membranes were washed three times with TBS-T and reacted with serum samples from patients with PCM or with serum samples from NC diluted 1:200 in a solution of 1% gelatin in TBS-T, for 2 h at 37°C. After the incubation, the membranes were washed five times and incubated with alkaline phosphatase-conjugated goat anti-human immunoglobulin antibody (isotyped M, G and A – whole molecule; Merck Millipore, USA), diluted 1:10,000 in a solution of 1% gelatin in TBS-T, for 1.5 hours at 37°C. The reaction was developed with BCIP-NBT (5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium; Sigma-Aldrich), according to the manufacturer's instructions.
3
Protein and DNA Marker Acquisition Protocol
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For the protein standards, we obtained the following from GE Healthcare (Chicago, IL, USA): PageRuler™ Plus Prestained Protein Ladder (catalog number 26619), LMW SDS Marker Kit (catalog number 17044601), and Coomassie™ Brilliant Blue (catalog number 20279). The DNA markers were acquired from Thermo Fisher Scientific (Waltham, MA, USA) and GE Healthcare Bio-Sciences AB (Piscataway, NJ, USA). The protease inhibitor used in the study was Protease Inhibitor Cocktail Tablets, EDTA-Free (catalog number S8820), obtained from Sigma-Aldrich (St. Louis, MO, USA). Sephacryl S300 HR and all other necessary chemicals were also acquired from Sigma-Aldrich (St. Louis, MO, USA).
4
SDS-PAGE Analysis of Digestion Process
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Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) method, according to Laemmli [86 (link)], using a ‘Mini-PROTEAN’ electrophoresis kit (Bio-Rad, USA), was used to monitor the digestion process. Analyses were conducted with a 12% separating gel, a 4% stacking gel, and an LMW-SDS Marker Kit (GE Healthcare Life Sciences, Chicago, IL, USA). The gels obtained were scanned (ImageScaner III, GE Healthcare, Great Britain), and the resulting images were analyzed.
5
Protein Extraction and Quantification Protocol
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Protein extraction was performed following the protocol described by Link et al. [106 (link)] to avoid vitellogenin sample contamination.
Protein quantification was carried out by densitometry analysis following SDS-PAGE. For that purpose, samples were run in precast gels (NuPAGE™ 4 to 12%, Bis-Tris, 1.0 mm, Mini Protein Gel, 10-well, Invitrogen). The LMW-SDS Marker Kit (GE Healthcare) was used as a standard. After staining with colloidal Coomassie blue, gel images were digitized with a UMAX Power-Look 1120 scanner and LabScan 5.0 software (GE Healthcare). Quantification was performed using ImageQuant TL software (v8.1), a 1D analysis module.
Twenty micrograms of protein extracted from each sample was run on a 3-cm-long SDS-PAGE gel. Each lane was excised into 3 fragments, transferred to Eppendorf tubes and processed independently. Proteins were in-gel reduced and alkylated by incubation with 10 mM DTT for 1 h at 56°C followed by incubation with 50 mM iodoacetamide for 45 min at room temperature. In-gel protein digestion was performed by incubation with 2 μg of trypsin (sequence grade, Promega) in 50 mM ammonium bicarbonate overnight at 37°C. Peptides were extracted from gel pieces by the addition of 60% acetonitrile/0.1% formic acid with vigorous agitation followed by vacuum drying and resuspension in 0.1% formic acid. Samples were centrifuged at 16,000xg for 30 min at 4°C.
Protein quantification was carried out by densitometry analysis following SDS-PAGE. For that purpose, samples were run in precast gels (NuPAGE™ 4 to 12%, Bis-Tris, 1.0 mm, Mini Protein Gel, 10-well, Invitrogen). The LMW-SDS Marker Kit (GE Healthcare) was used as a standard. After staining with colloidal Coomassie blue, gel images were digitized with a UMAX Power-Look 1120 scanner and LabScan 5.0 software (GE Healthcare). Quantification was performed using ImageQuant TL software (v8.1), a 1D analysis module.
Twenty micrograms of protein extracted from each sample was run on a 3-cm-long SDS-PAGE gel. Each lane was excised into 3 fragments, transferred to Eppendorf tubes and processed independently. Proteins were in-gel reduced and alkylated by incubation with 10 mM DTT for 1 h at 56°C followed by incubation with 50 mM iodoacetamide for 45 min at room temperature. In-gel protein digestion was performed by incubation with 2 μg of trypsin (sequence grade, Promega) in 50 mM ammonium bicarbonate overnight at 37°C. Peptides were extracted from gel pieces by the addition of 60% acetonitrile/0.1% formic acid with vigorous agitation followed by vacuum drying and resuspension in 0.1% formic acid. Samples were centrifuged at 16,000xg for 30 min at 4°C.
6
Mycobacterium tuberculosis PknG Null Mutant
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The Mycobacterium tuberculosis pknG null mutant strain (ΔpknG) was kindly provided by Josef Av-Gay (18) (link). Wild-type M. tuberculosis H37Rv (WT) and ΔpknG strains were cultured in 50 mL of Middlebrook 7H9 medium supplemented with 0.05% Tween® 80, albumin-dextrose and asparagine (BD Biosciences) until early-logarithmic phase as previously described (19) (link). Mycobacterial cells were washed with PBS buffer, resuspended in minimum medium supplemented with 10 mM asparagine and cultured for five additional days. Cells were harvested by centrifugation (3000 g for 10 min at 4 °C), washed in PBS buffer containing the Complete EDTA-free Protease Inhibitor Cocktail (Roche) in the amount recommended by the supplier. To prepare whole cell lysates, an equal amount of acid-washed glass beads (≤ 106 µm, Sigma) was added to the cell suspension and the system was vortexed at maximum speed for 10 min. Cell debris and beads were removed by centrifugation at 1,000 g for 5 min at 4 °C and lysates filtered through 0.22 μm PVDF membranes and stored at -80 °C for further analysis. Protein quantification was performed by gel densitometry measurements using the 1D analysis module of the ImageQuant TL software (v8.1) and the LMW-SDS Marker Kit (GE Healthcare) as standard. Protein extracts for the WT and ΔpknG M. tuberculosis strains were prepared in triplicate.
7
Immunostaining of cTnI Fragments in AMI Patients
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Proteins were separated with a 15%-22.5% gradient SDS-PAGE according to Laemmli (10 ) under reducing conditions by a Hoefer SE280 electrophoresis unit (gel size 10 ϫ 12 cm), blotted onto an Amersham HyBond-ECL nitrocellulose membrane (GE Healthcare), immunostained by anti-cTnI mAbs conjugated with HRP, and visualized with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) on the ChemiDoc Touch Imaging System (Bio-Rad) (for details, see the online Data Supplement). The apparent molecular masses of the separated proteins were determined with an LMW-SDS Marker Kit (GE Healthcare).
To determine the approximate borders of cTnI fragments, proteins immunoprecipitated from serum samples of AMI patients (n ϭ 9) were applied on 1-well electrophoresis gels and after separation were blotted onto a nitrocellulose membrane. Then, the membrane was cut into strips, and each strip was immunostained with 1 of the 15 mAbs specific to the different epitopes of cTnI.
To determine the approximate borders of cTnI fragments, proteins immunoprecipitated from serum samples of AMI patients (n ϭ 9) were applied on 1-well electrophoresis gels and after separation were blotted onto a nitrocellulose membrane. Then, the membrane was cut into strips, and each strip was immunostained with 1 of the 15 mAbs specific to the different epitopes of cTnI.
8
Characterization of a Lectin's Properties
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The effects of pH, temperature, divalent cations, and EDTA on HA were determined as described by Sampaio et al. (1998) . Molecular mass of the lectin was estimated by SDS-PAGE, as described by Laemmli (1970) . LMW-SDS marker kit (GE Healthcare, IL, USA) was used as standard.
Native molecular mass was estimated by size exclusion chromatography (SEC) on BioSuite 250 HR SEC column (0.78 × 30 cm, 5-μm particle size, Waters Corp., MA, USA), coupled to an Acquity UPLC system (Waters Corp., MA, USA), which had been equilibrated with Tris-HCl 20 mM, pH 7.2, containing NaCl 500 mM. The column was previously calibrated with a mixture of standard proteins: conalbumin (75 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa), ribonuclease A (14 kDa), and aprotinin (6.5 kDa).
Native molecular mass was estimated by size exclusion chromatography (SEC) on BioSuite 250 HR SEC column (0.78 × 30 cm, 5-μm particle size, Waters Corp., MA, USA), coupled to an Acquity UPLC system (Waters Corp., MA, USA), which had been equilibrated with Tris-HCl 20 mM, pH 7.2, containing NaCl 500 mM. The column was previously calibrated with a mixture of standard proteins: conalbumin (75 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa), ribonuclease A (14 kDa), and aprotinin (6.5 kDa).
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