The sequences for the Mtb proteins Rv1196 and Rv0125 were obtained from publicly available databases. Peptide libraries were generated using a commercially available peptide library design tool (PEPscreen, Millipore Sigma, Merck, Darmstadt, Germany) with peptide lengths of 20 amino acids (a.a.) and an overlap of 10 a.a. Individual peptides were ordered from Genscript and were resuspended in DMSO (with a calculated final concentration of no greater than 10%) and sterile 1× PBS, according to their molecular weights, to obtain a stock concentration of 10 mM. The stock concentrations were diluted using sterile 1× PBS to 1 mg/mL working concentrations. Peptides were used at 1 to 4 μg/mL, according to assay standards.
Pepscreen
Manufactured by Merck Group
Sourced in United States
PEPscreen is a laboratory equipment product offered by Merck Group. It is designed for high-throughput screening of peptides. The core function of PEPscreen is to facilitate the rapid and efficient evaluation of large peptide libraries or collections.
Automatically generated - may contain errors
Lab products found in correlation
Imark microplate reader, by Bio-Rad (6 mentions)
Nunc maxisorp 96 well immunoplates, by Thermo Fisher Scientific (6 mentions)
Superblock t20 pbs blocking buffer, by Thermo Fisher Scientific (5 mentions)
1 step ultra tmb elisa, by Thermo Fisher Scientific (5 mentions)
Peroxidase conjugated anti mouse igg, by Agilent Technologies (4 mentions)
Elisa pod substrate tmb kit, by Nacalai Tesque (2 mentions)
Infinite m200 pro, by Tecan (1 mentions)
Canto 2, by BD (1 mentions)
Peroxidase conjugated anti rat immunoglobulins, by Merck Group (1 mentions)
Anti human ifnγ antibody, by Mabtech (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Multiscreen hts filter, by Merck Group (1 mentions)
Anti ifn γ antibody clone 7 b6 1, by Mabtech (1 mentions)
Elisa microplate reader, by Agilent Technologies (1 mentions)
Bcip nbt, by Mabtech (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Biotinylated anti ifnγ antibody clone 7 b6 1, by Mabtech (1 mentions)
Streptavidin alp, by Mabtech (1 mentions)
Maxisorp 96 well immunoplates, by Thermo Fisher Scientific (1 mentions)
Elispot kit, by Mabtech (1 mentions)
21 protocols using pepscreen
1
Peptide Screening for Mycobacterium tuberculosis Proteins
2
Fluorogenic Protease Substrate Synthesis and Assay
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Fluorogenic protease substrates were synthesized using Millipore-Sigma’s customer peptide library service PepScreen (LQx) or by CPC (PPx, GzmB, NE, CatK) (table S1). Recombinant proteases were purchased from Enzo Life Sciences, R&D Systems, and Haematologic Technologies. For recombinant protease assays, fluorogenic substrates LQx (60 μM final concentration) or PPx, GzmB, NE, and CatK (1 μM final concentration) were incubated in 30-μl final volume in appropriate enzyme buffer, according to manufacturer specifications, with 12.5 nM recombinant enzyme at 37°C (fig. S2B). Proteolytic cleavage of substrates was quantified by increases in fluorescence over time by a fluorimeter (Tecan Infinite M200 Pro). Enzyme cleavage rates were quantified as relative fluorescence increase over time normalized to fluorescence before addition of protease. Hierarchical clustering was performed in GENE-E (https://software.broadinstitute.org/GENE-E/ , Broad Institute), using z-scored fluorescence fold changes at 60 min.
3
ATRX Peptide Immobilization and AMab-6 Binding
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Synthesized ATRX peptides using PEPScreen (Sigma-Aldrich Corp., St. Louis, MO, USA) were immobilized on Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 10 μg/mL for 30 min at 37 °C. After blocking with SuperBlock T20 (PBS) Blocking Buffer (Thermo Fisher Scientific Inc.), the plates were incubated with 10 μg/mL purified AMab-6 followed by a 1:2000 dilution of peroxidase-conjugated anti-mouse IgG (Agilent Technologies Inc.). The enzymatic reaction was conducted using 1-Step Ultra TMB-ELISA (Thermo Fisher Scientific Inc.). Optical density was measured at 655 nm using an iMark Microplate Reader (Bio-Rad Laboratories, Inc., Berkeley, CA, USA). These reactions were performed at 37 °C using a total sample volume of 50–100 μL.
4
Quantifying Antigen-Specific CD8+ T Cell Cytotoxicity
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The lytic activity of SIINFEKL-specific CD8+ T cells in PbAAma1OVA-infected animals was determined with an in vivo cytotoxicity assay on day 6 p.i. as described before (42 (link)). Briefly, splenocytes from syngenic donor mice were pulsed with 1 μM of the ovalbumin–derived specific H-2kb peptide SIINFEKL (PEPscreen/ Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 37°C and subsequently with 1 μM of 5(6)-Carboxyfluorescein diacetate N-succinimidyl ester (CFSE, Sigma-Aldrich, St. Louis, MO, USA) for 15 min (CFSEhigh, specific target cells). Reference cells were not pulsed with peptide and labeled with 0.1 μM CFSE for 15 min (CFSElow, reference cells). Cells were washed with 1 X PBS (PAA, Cölbe, Germany) and the cell number was determined. The cell populations were mixed at a 1:1 ratio (CFSEhigh/CFSElow). Each recipient received 5x106 cells of each population diluted in 0.9% NaCl (Braun, Melsungen, Germany) into the tail vein on day 5 p.i.. Mice were sacrificed 18 h later on day 6 p.i. and spleens were isolated to prepare single-cell suspensions as described above. Lysis of peptide-loaded cells was quantified by measuring the ratio of CFSEhigh/ CFSElow cells via flow cytometry (Canto II, BD Biosciences). The percentage of specific lysis, termed S8L-specific lysis, was calculated using the following equation: 100 - [(CFSEhigh/CFSElow) immunized/(CFSEhigh/CFSElow) naïve] x 100.
5
Mapping mCCR9 Peptide Binding Epitopes
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The mCCR9 peptides, such as wild type (WT), 19 of 1× alanine (1× Ala)-substituted peptides (Table 1 ), and 18 of 2× alanine (2× Ala)-substituted peptides (Table 2 ), were synthesized using PEPscreen (Sigma-Aldrich Corp., St. Louis, MO, USA). Each peptide was immobilized on Nunc Maxisorp 96-well immuno plates (Thermo Fisher Scientific, Inc.) at a concentration of 1 μg/mL for 30 min at 37 °C. As a negative control, no peptide was immobilized on the immuno plates. After washing with phosphate-buffered saline containing 0.05% Tween20 (PBST), the wells were blocked with 1% bovine serum albumin containing PBST for 30 min at 37 °C. The plates were then incubated with C9Mab-24 (1 μg/mL), followed by a 1:20,000 dilution of peroxidase-conjugated anti-rat immunoglobulins (Sigma-Aldrich Corp.). Enzymatic reactions were performed using an ELISA POD Substrate TMB Kit (Nacalai Tesque, Inc.). Optical density was detected at 655 nm using an iMark microplate reader (Bio-Rad Laboratories, Inc., Berkeley, CA, USA).
6
Generating Overlapping Peptides for CAR T-Cell Screening
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Individual 15-mer peptides with 11 amino acid overlapping sequences were generated against amino acids 8–279 of the 11D5-3-CD828Z CAR11 (link) (PEPScreen, Sigma Aldrich). The 15-mers cover the light chain, linker, and heavy chain of the 11D5-3 scFv sequence. Individual peptides were dissolved in dimethyl sulfoxide to a concentration of 20 mg/mL. For rapid screening, 13 pools, each consisting of five peptides, were generated.
7
IFNγ ELISpot Assay for Anti-TetR Responses
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Anti-TetR cellular immune responses were evaluated with an IFNγ ELISpot assay using frozen PBMC isolated at >18 months postinjection. The assay was performed using an overlapping peptide library covering the rtTA sequence (15 per 10 mers, Pepscreen, Sigma) split in five peptide pools. Briefly, the method consisted in plating 2 × 105 cells in human anti-IFNγ antibody (MabTech, France) precoated MultiScreenHTS filter plates with polyvinyldiene difluoride membrane (PVDF, Millipore, France). Cells were restimulated with rtTA-derived pool peptides at a final concentration of 10 μg/ml. Medium alone and an irrelevant pool of peptides served as negative controls, while cells stimulated with phorbol-12-myristate-13-acetate (PMA, 10 ng/ml, Sigma)/calcium ionophore A23187 Mi Calcium Salt (ionomycin, 250 ng/ml, Sigma) served as a positive control. After incubation with a biotinylated anti-IFN-γ antibody (clone 7-B6-1, MabTech, France) and ExtrAvidin alkalin phosphatase (Sigma-Aldrich), enzymatic reaction was revealed using NBT/BCIP (Pierce, Thermo Scientific). Spot number was determined using an ELISpot reader ELR07 (AID, Straßberg, Germany) and analyzed with AID ELISpot Reader Software V7.0 (AID, Germany). Responses were considered positive when the number of spot-forming colonies per million cells was >50 and at least threefold higher than the peptide pool negative control (C−).
8
Synthesis and Characterization of FV Peptides
9
Synthesis of mCCR6 Peptide Analogs
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The mCCR6 (Accession No.: NM_001190333.1) peptide (1-MNSTESYFGTDDYDNTEYYS-20) and 1× alanine residue-substituted peptides (Table 1 ) were synthesized utilizing PEPscreen (Sigma-Aldrich Corp.).
10
PDPN Peptide Immobilization and Antibody Detection
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Synthesized rPDPN peptides using PEPscreen (Sigma-Aldrich Corp., St. Louis, MO) were immobilized on Nunc MaxiSorp 96-well immunoplates (Thermo Fisher Scientific, Inc.) at 10 μg/mL for 30 minutes at 37°C. After blocking with Superblock T20 (PBS) blocking buffer (Thermo Fisher Scientific, Inc.), the plates were incubated with purified PMab-2 (10 μg/mL), followed by a 1:2000 dilution of peroxidase-conjugated anti-mouse IgG (Agilent Technologies, Inc.). The enzymatic reaction was conducted using 1-Step Ultra TMB-ELISA (Thermo Fisher Scientific, Inc.). Optical density was measured at 655 nm using an iMark Microplate Reader (Bio-Rad Laboratories, Inc., Berkeley, CA). These reactions were performed at 37°C with a total sample volume of 50–100 μL.
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