Immobilized pepsin

Manufactured by Thermo Fisher Scientific

Sourced in United States

Immobilized pepsin is a laboratory reagent used in the preparation and analysis of protein samples. It consists of the digestive enzyme pepsin, which has been covalently bound to a solid support matrix. This immobilization allows for the controlled enzymatic digestion of proteins, which is a common step in various analytical and research procedures.

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Lab products found in correlation

Thermolysin, by Promega (1 mentions) Arg c, by Promega (1 mentions) Trypsin, by Promega (1 mentions) Rituximab, by Genentech (1 mentions) Anti cd45 fitc, by BD (1 mentions) Anti cd86 pe, by Thermo Fisher Scientific (1 mentions) Anti igd fitc, by BD (1 mentions) Anti cd80 fitc, by Thermo Fisher Scientific (1 mentions) Anti cd19 apc, by BD (1 mentions) Anti cd27 pe, by BD (1 mentions) Rituximab, by Thermo Fisher Scientific (1 mentions) Recombinant human il 4, by Thermo Fisher Scientific (1 mentions) Protein a sepharose beads, by GE Healthcare (1 mentions) Imiquimod, by InvivoGen (1 mentions) Pepsin, by Thermo Fisher Scientific (1 mentions) Nab protein a plus column, by Thermo Fisher Scientific (1 mentions) Protein g chromatography, by GE Healthcare (1 mentions) Protein a affinity column, by GE Healthcare (1 mentions)

5 protocols using immobilized pepsin

Proteomic analysis by HPLC fractions

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Samples of each HPLC-separated fraction were digested with four different endoproteases: trypsin, Arg-C, thermolysin (Promega, USA) and immobilized pepsin (Thermo Scientific, USA). The digestions were performed according to standard protocols provided by manufacturers.

Kotliński M., Rutowicz K., Kniżewski Ł., Palusiński A., Olędzki J., Fogtman A., Rubel T., Koblowska M., Dadlez M., Ginalski K, & Jerzmanowski A. (2016). Histone H1 Variants in Arabidopsis Are Subject to Numerous Post-Translational Modifications, Both Conserved and Previously Unknown in Histones, Suggesting Complex Functions of H1 in Plants. PLoS ONE, 11(1), e0147908.

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Multiparametric Flow Cytometry Analysis

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FITC anti-CD45, APC anti-CD19, FITC anti-IgD and PE anti-CD27 were purchased from BD Biosciences. PE anti-IL-6, FITC anti-CD80, PE anti-CD86, PE anti-BR3, and PE anti-CD40 were purchased from eBiosciences. LEAF (low endotoxin, azide-free) anti-CD40 was purchased from BioLegend. Rituximab (Genentech) was obtained through the hospital pharmacy. Rituximab F(ab’)2 fragments were created by treating RTX with immobilized pepsin (Thermo Scientific) per manufacturer's instructions, followed by removal of Fc fragments using protein A Sepharose beads (GE Healthcare), and purity (>99%) was confirmed by SDSPAGE. Recombinant human IL-4 was purchased from eBiosciences. Imiquimod and CpG (ODN 2006) were purchased from InvivoGen. Carboxyflourescein succinimidyl ester (CellTrace CFSE) was purchased from Life Technologies.

Jones J.D., Hamilton B.J., Skopelja S, & Rigby W.F. (2014). Rituximab induces Interleukin-6 production by human B cells. Arthritis & rheumatology (Hoboken, N.J.), 66(11), 2938-2946.

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Virus Particle Infectivity Assay

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Cultures of the chronically infected strain or the isogenic uninfected strain were grown to an optical density of 0.15 to 0.20 and then filtered through a 0.22-μm PES filter. Supernatants were then placed in boiling water for 30 min. Supernatant samples from before and after boiling were tested for infectious particles by plaque assay as described above. When pepsin was used, the treatment of the supernatants was completed with immobilized pepsin (Thermo Scientific catalog number 20343) according to the manufacturer’s instructions and incubated at 37°C overnight in a rotator. Strains tested were grown to mid-log phase and then diluted to an OD₆₀₀ of 0.03 in fresh medium, the boiled supernatants, or the unboiled supernatants. At 24, 48, and 72 h, cultures were centrifuged, and cells were resuspended in fresh doses of medium or the supernatants. Every 24 h, cell viability was tested by spotting 10-μl cell dilutions onto DTU plates.

DeWerff S.J., Bautista M.A., Pauly M., Zhang C, & Whitaker R.J. (2020). Killer Archaea: Virus-Mediated Antagonism to CRISPR-Immune Populations Results in Emergent Virus-Host Mutualism. mBio, 11(2), e00404-20.

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Antibody Digestion and Purification Protocol

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K9.361 and 2.4G2 mAbs were digested for 7.5 hours at 37°C with immobilized pepsin (Thermo Scientific) while being rotated and then purified with the NAb Protein A Plus column (Thermo Scientific) and concentrated with centrifugal filter devices (Amicon, EMD Millipore, Norwood, Ohio). Aliquots were compared with undigested intact mAb under nonreducing conditions by using SDS-PAGE with 7% polyacrylamide gels (NuPAGE Tris-Acetate; Life Technologies, Grand Island, NY) and lacked detectable intact IgG (see Fig E1 in this article’s Online Repository at www.jacionline.org ).^{25 (link)}

Clay C.D., Strait R.T., Mahler A., Khodoun M.V, & Finkelman F.D. (2017). Anti-FcγRIIB mAb suppresses murine IgG-dependent anaphylaxis by Fc domain targeting of FcγRIII. The Journal of allergy and clinical immunology, 141(4), 1373-1381.e5.

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Purification and characterization of IgG

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Blood or plasma exchange fluid was obtained from patients with ANCA vasculitis or healthy controls and plasma stored at –80°C. Fibrinogen was precipitated by adding 18 g/100 ml of sodium chloride and IgG was purified with protein G chromatography (GE Healthcare). The endotoxin concentration in the final IgG preparations was measured by Lonza using a LAL kinetic chromogenic assay. For all 30 polyclonal IgGs used, the endotoxin was less than 0.05 eU/250 μg IgG. The concentration of IgG used for all neutrophil and monocyte assays was 250 μg/ml. To generate F(ab)₂ fragments, whole IgG was buffer exchanged using PD10 columns into 20 mM sodium acetate and digested using immobilized pepsin (Thermo Fisher Scientific). The F(ab)₂ fragments were separated from undigested IgG using protein A affinity columns (GE Healthcare). SDS-PAGE demonstrated the purity of whole IgG and the F(ab)₂ fragments. Before all assays, IgG preparations were centrifuged at 16,000 g for 30 minutes at 4°C to remove aggregates. In mechanistic experiments where smaller numbers of IgG samples were used, these were taken from this full set of 30 samples.

Popat R.J., Hakki S., Thakker A., Coughlan A.M., Watson J., Little M.A., Spickett C.M., Lavender P., Afzali B., Kemper C, & Robson M.G. (2017). Anti-myeloperoxidase antibodies attenuate the monocyte response to LPS and shape macrophage development. JCI Insight, 2(2), e87379.

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