Corneas and submandibular lymph nodes (MLN) were homogenized with a Tissuemiser (Fisher Scientific, Pittsburgh, PA, USA) in the presence of 1× Calbiochem protease inhibitor cocktail set I (EMD Millipore, Billerica, MA, USA ) in 1× PBS. The tissue homogenates were centrifuged at 10,000 × g for 3 minutes at 4°C. Supernatants were collected and assessed for VEGF-A content using a standard ELISA kit (R&D Systems, Minneapolis, MN, USA) or IL-2, IL-7, IL-12 p70, and IL-15 content using a multiplex suspension array kit (EMD Millipore). VEGF-A ELISA plates were analyzed using a CLARIO star microplate reader (BMG LABTECH, Cary, NC, USA). The luminex x-map bead-based multiplex assays were analyzed using a Bio-plex 200 system (Bio-Rad, Hercules, CA, USA).
Calbiochem protease inhibitor cocktail set 1
Manufactured by Merck Group
Sourced in United States
Calbiochem protease inhibitor cocktail set I is a laboratory product designed to inhibit a wide range of proteases. It is a premixed solution containing multiple protease inhibitors formulated to prevent the degradation of proteins during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Tissuemiser, by Thermo Fisher Scientific (4 mentions)
Multiplex suspension array kit, by Merck Group (2 mentions)
Standard elisa kit, by R&D Systems (2 mentions)
Bio plex 200 system, by Bio-Rad (2 mentions)
Clariostar microplate reader, by BMG Labtech (2 mentions)
Luminex instrument, by Bio-Rad (2 mentions)
Quantigene 2.0 multiplex kit, by Thermo Fisher Scientific (2 mentions)
T per tissue protein extraction reagent, by Thermo Fisher Scientific (2 mentions)
T per, by Thermo Fisher Scientific (2 mentions)
Hstcmag28spmx13, by Merck Group (2 mentions)
Prism 7, by GraphPad (2 mentions)
Tissuelyser lt, by Qiagen (2 mentions)
Protein a g agarose bead, by Roche (2 mentions)
Immobilon p pvdf membrane, by Merck Group (1 mentions)
9 protocols using calbiochem protease inhibitor cocktail set 1
1
Quantification of Immune Factors in Corneal and Lymph Node Tissues
2
Quantification of Immune Factors in Corneal and Lymph Node Tissues
3
Western Blot Analysis of GvpC Protein
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Cells were grown in LB as indicated above for aerated conditions, collected at 16 h, normalized to 2.0 OD600, pelleted at 8000 g and 4°C, and resuspended in 1.25 ml of CHAPS lysis buffer containing 1X Calbiochem protease inhibitor cocktail set I (Merck) (Coulthurst et al.,2006 ). The lysis solution was kept on ice and sonicated for 3 cycles × 20 s. Cell debris and insoluble material was pelleted at 13 000 g and 4°C. Protein samples were separated using 15% acrylamide SDS gels. Proteins were transferred to an Immobilon‐P PVDF membrane (Merck), washed three times for 5 min with 0.1% (v/v) Tween 20 in phosphate‐buffered saline (PBS), and blotted in 5% (w/v) milk in Tween 20‐PBS (blocking solution) with rabbit GvpC antibody (1:30 000 antibody to blocking solution volume ratio) for 1 h and goat IgG (1:30 000 IgG to blocking solution volume ratio) for 40 min. The GvpC antibody was raised against the MAQLKNIDDSHES peptide, immunized in rabbits (BioGenes GmbH) and was pre‐absorbed to whole protein precipitates from a ΔgvpC strain of S39006 before usage (Tashiro et al.,2016 ).
4
Multiplex Gene and Protein Profiling
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For gene array, corneas were processed using a magnetic bead-based Quantigene 2.0 multiplex kit (Affymetrix). Briefly, samples were lysed to extract RNA and incubated with specific target probes overnight. Signals were amplified with a hybridization technique, and detected using a luminex instrument (Biorad) after adding streptavidin with phycoerythrin as a substrate. For protein array, tissues were homogenized with a Tissuemiser (Fisher Scientific) in Tissue Protein Extraction Reagent (T-PER, Fisher Scientific) in the presence of 1x Calbiochem protease inhibitor cocktail set I (Millipore). Homogenates were centrifuged in a microcentrifuge at 10,000×g for 90 sec at 4° C, and the supernatants were evaluated for analyte content by multiplex (Millipore) or ELISA (R&D) assays.
5
Multiplex Gene and Protein Profiling
6
Protein Expression Analysis in 3D Constructs
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3D constructs were washed with 1 mL 1X PBS and then, using a spatula, were gently scraped off of the membrane and placed in a 2 mL tube containing a 6.35 mm metal ball, T-PER (ThermoScientific, Ref. #78510), and Calbiochem protease inhibitor cocktail set I (Millipore Sigma, Cat. #539131–1VL). Samples were then homogenized using a Qiagen TissueLyser LT at 50 oscillations/second for 30 s (x2). Following 30 min incubation at 4 °C, samples were centrifuged at 12,000 RPM for 15 min at 4 °C. Supernatants were collected and stored at − 80 °C until the time of the experiment. A bead-based multiplex assay for protein detection was used to quantify expression of GM-CSF, IFN-ɣ, IL-10, IL-13, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-12 (p70), and TNF- a (MilliporeSigma, HSTCMAG28SPMX13). Data were graphed in GraphPad Prism 7 and statistics were determined using Welch’s t-test where p ≤ 0.05*, p ≤ 0.01**, p ≤ 0.001***, and p ≤ 0.0001****. Samples with values that were too low to be extrapolated from the standard curve were assigned a value of 0.01.
7
Protein Expression Analysis in 3D Constructs
8
LPS-Induced Signaling Pathway Analysis
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HT-29 cells were stimulated with LPS (200 ng/ml) for 4 hr in medium without FBS. After the stimulation, cells were washed with ice-cold PBS and lysed with ice-cold IP buffer (10 mM HEPES pH 7.4; 150 mM NaCl, 1% Triton X-100, plus protease inhibitor cocktail Set I-Calbiochem 1:100 (Cat# 539131, Merck Millipore), 1 mM Na3VO4 and 1 mM NaF). After 30 min on ice, samples were centrifuged at 14,000 rpm for 10 min. Protein A/G-Agarose beads (Cat# 11 134 515 001/11 243 233 001, Roche), lysates and either c-Jun, ERK3 or Normal Rabbit IgG antibody were incubated for 2 hr at 4°C with rotating. After the incubation, beads were washed with IP buffer and analyzed by immunoblot.
9
Immunoprecipitation of ARP3 Complex
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HMECs were seeded in 6-well plate at an initial density of 2 × 105 cells per well and cultured until 80% confluent. For the immunoprecipitation (IP) of the endogenous ARP3, cells were washed with ice-cold PBS and lysed with ice-cold IP buffer (10 mM HEPES pH 7.4; 150 mM NaCl, 1% Triton X-100, plus protease inhibitor cocktail Set I-Calbiochem 1:100 [Cat# 539131, Merck Millipore], 1 mM Na3VO4, and 1mM NaF). Cell lysates were incubated with Protein A/G-Agarose beads (Cat# 11 134 515 001/11 243 233 001, Roche), and either ARP3 or normal rabbit IgG antibody for 2 hr at 4°C with rotating. After the incubation, beads were washed with IP buffer and analyzed by immunoblot. Levels of the immunoprecipitated ARP3 as well as the co-immunoprecipitation of ARP2 and ERK3 were assessed. Total cell lysates were used as a control.
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