Binding ability of inhibitors to cell membranes was determined by flow cytometry. Briefly, a peptide or protein inhibitor was added to TZM-bl cells (1 × 106) and incubated at 4°C for 1 h. After washing twice with fluorescence activated cell sorting (FACS) buffer [PBS supplemented with 0.5% bovine serum albumin and 2 mM ethylene diamine tetraacetic acid (EDTA)], cells were incubated with a mouse anti-His tag antibody (Sigma) at 1:200 dilution for 1 h at 4°C. Then, the cells were washed twice and incubated with Alexa Fluor 488 rabbit anti-mouse IgG antibody (Invitrogen, Carlsbad, CA, USA) for 1 h at 4°C. Subsequently, cells were stained with allophycocyanin–conjugated mouse anti-human CD4 antibody (BD Biosciences, Franklin Lakes, NJ, USA) for 1 h at 4°C and then resuspended by FACS buffer containing 4% formaldehyde. FACS analysis was conducted with FACSCantoII instrument.
Mouse anti his tag antibody
Manufactured by Merck Group
Sourced in United States
The Mouse anti-His tag antibody is a laboratory reagent used to detect and purify proteins that have been engineered with a histidine (His) tag. The antibody recognizes and binds to the His tag, allowing for the identification and isolation of the tagged protein from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Ultrafiltration tube, by Merck Group (1 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Freestyle 293 expression media, by Thermo Fisher Scientific (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Anti m13 hrp, by Merck Group (1 mentions)
7 protocols using mouse anti his tag antibody
1
Membrane-Binding Affinity Assay by Flow Cytometry
2
Western Blot Analysis of Transgene Expression
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To detect the expression of transgenes in transduced TZM-bl cells, 3×106 cells were plated in a P-100 dish with 10 ml complete DMEM containing 10% FBS and incubated at 37°C in 5% CO2. After incubation for 24 h, the culture media were replaced with Freestyle 293 expression media (Invitrogen) and cultured for additional 48 h. Then, the cells and supernatants were harvested, respectively. Cells were lysed with ice-cold RIPA lysis buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS; Invitrogen) containing protease inhibitor cocktail (Roche). Proteins in supernatant were concentrated by ultrafiltration tube (15 ml, 10 kDa; Millipore). Samples were separated in 10% SDS-PAGE, transferred to a nitrocellulose membrane, followed by staining with mouse anti-His tag antibody (Sigma).
3
Western Blot Analysis of His-Tagged Proteins
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Protein extracts were fractionated by SDS-PAGE and transferred into a nitrocellulose membrane according to the manufacturer's protocols (Bio-Rad®). After incubation with 1% skimmed milk in TBST for 2 h, the membrane was washed three times with TBST and incubated with mouse anti-His-tag antibody (Sigma-Aldrich®, 1:5000), at room temperature for 2 h. For HRP labeled western blots, membranes were washed three times with TBST-Low salt (10 mM NaCl) and incubated with 2.5 mg/mL HRP conjugated SA-TRIM21 or HRP-conjugated anti-mouse IgG for 2 h. Blots were washed with TBST-Low salt (10 mM NaCl) three times and developed with ECL-Luminol (Scienco Biotech) according to the manufacturer’s protocols.
4
Protein Purity and Size Analysis
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To determine the purity and molecular size of purified iMabSC and 2P23-iMab, the protein samples were loaded onto a 10% Sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) separating gel with equal mass and the gel was stained with Coomassie brilliant blue. The recombinant inhibitors were verified by Western blotting assay. Briefly, equal amounts of the purified proteins were separated by 10% SDS-PAGE gel and transferred to a nitrocellulose membrane, which was then blocked for 1 h with a 5% (wt/vol) solution of nonfat dry milk in Tris-buffered saline–Tween 20 (TBST, pH 7.4) at room temperature. The membrane was incubated with a mouse anti-His tag antibody (Sigma) at 1:3,000 dilution or 2P23 peptide-specific MAb 5F7 (4 μg/ml) overnight at 4°C. After washing three times with TBST, the membrane was incubated with IRDye 680RD-conjugated Goat anti-Mouse immunoglobulin G (IgG) antibody (Rockland, Philadelphia, Pennsylvania, USA) at 1:20,000 dilution at room temperature for 1 h. Imaging was performed by the LI-COR Odyssey imaging system (LI-COR Biosciences, Lincoln, NE, USA).
5
PPRV Vaccine and Nanobody Development
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The live attenuated PPRV/N/75/1 vaccine strain was outsourced from Botswana Vaccine Institute, Botswana for immunisation of the alpaca. Whole killed PPRV antigen mixture generated from completely inactivated PPRV was obtained from The Pirbright Institute, United Kingdom for affinity selection of PPRV-reactive nanobodies. For enzyme-linked immunosorbent assay (ELISA) tests, rabbit anti-camel VHH antibody, goat anti-rabbit-horseradish peroxide (HRP, BioRad, Hercules, CA, USA), anti-M13-HRP, mouse anti-His tag antibody, and goat anti-mouse alkaline phosphatases (Sigma-Aldrich, St. Louis, MO, USA) were all provided by Vrije Universiteit Brussel, Brussels, Belgium and all were used according to the manufacturer’s instructions.
6
Characterization of Recombinant TYMS Protein
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After the purification, Coomassie brilliant blue was used to examine the purity of the proteins. Purified proteins were series diluted (0-, 2-, 4-, 8-, 16-, 32-, 64-, 128-, 256-fold) and then incubated with a loading buffer with or without β-mercaptoethanol at 37 °C or 100 °C for 10 min. Then, these sample were subjected to SDS-PAGE to determine the protein purity and oligomeric form. After that, 10 µM TYMS was incubated with 2 mM dUMP and 2 mM mTHF in a 40 µL reaction system at 37 °C and then subjected to SDS-PAGE to confirm the active or inactive state of TYMS.
Western blot was used to verify the protein expression, TYMSs were subjected to SDS-PAGE, the SDS gel was washed with a transfer buffer, and then the proteins were transferred from the gel onto a nitrocellulose membrane with a constant current of 250 mA for 2 h. The membrane was blocked with 1% nonfat milk powder in PBST (PBS containing 0.05% (v/v) Tween-20). Mouse anti-His-tag antibody (Sigma, St. Louis, MO, USA) was used as the primary antibody at a 1:5000 dilution in blocking solution. Goat-anti-mouse was the secondary antibody (Sigma), which was diluted at 1:3000, and protein bands were detected on photographic films using an enhanced chemiluminescent substrate.
Western blot was used to verify the protein expression, TYMSs were subjected to SDS-PAGE, the SDS gel was washed with a transfer buffer, and then the proteins were transferred from the gel onto a nitrocellulose membrane with a constant current of 250 mA for 2 h. The membrane was blocked with 1% nonfat milk powder in PBST (PBS containing 0.05% (v/v) Tween-20). Mouse anti-His-tag antibody (Sigma, St. Louis, MO, USA) was used as the primary antibody at a 1:5000 dilution in blocking solution. Goat-anti-mouse was the secondary antibody (Sigma), which was diluted at 1:3000, and protein bands were detected on photographic films using an enhanced chemiluminescent substrate.
7
Western Blot Protein Detection
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Protein extracts were fractionated by SDS-PAGE and transferred into a nitrocellulose membrane according to the manufacturer's protocols (Bio-Rad®). After incubation with 1% skimmed milk in TBST for 2 hours, the membrane was washed three times with TBST and incubated with mouse anti-His-tag antibody (Sigma Aldrich®, 1:5000), at room temperature for two h. For HRP labeled western blots, membranes were washed three times with TBST-Low salt (10 mM NaCl) and incubated with 2,5 mg/mL HRP conjugated SA-TRIM21 or HRP-conjugated anti-mouse IgG for two h. Blots were washed with TBST-Low salt (10 mM NaCl) three times and developed with ECL-Luminol (Scienco Biotech) according to the manufacturer's protocols.
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