Immunoprecipitation (IP) of FT2Fc from cell culture supernatant was performed as previously described with minor modifications [23 (link)]. Briefly, sheep-anti mouse IgG paramagnetic beads (Dynal, 11201D) were coupled with anti-His mAb (Invitrogen, 37–2900) in coating buffer (PBS plus 0.1% immunoglobulin-free BSA) for 2 h at RT on a rotating wheel. Three molar excess of His-tagged Fab83 were added. After 1 h incubation, beads were washed three times with coating buffer. Cell culture supernatant was diluted 1:1 in IP buffer (50 mM Tris-Cl, 75 nM NaCl, 1% Igepal, protease inhibitor mixture (Sigma, 11836153001), pH 7.4) plus 0.5% BSA and incubated with 50 μl of Fab83-anti-His antibody coupled beads. The same input was used for all conditions. IP was performed overnight at 4°C. After five washes with 50 mM Tris-Cl, 0.5% Igepal, 150 nM NaCl, 0.5% BSA, pH 7.4, elution of immunoprecipitated FT2Fc was performed by incubation with peptides (200 molar excess compared to Fab83) for 2 h at 4°C. The eluate was boiled in 4 x LDS for western blotting. After elution, the beads were boiled in 4 x LDS and the supernatant was investigated by western blotting.
Anti his mab
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Anti-His mAb is a monoclonal antibody that specifically recognizes the polyhistidine tag (His-tag) commonly used for protein purification and detection. This antibody can be used to detect and capture His-tagged recombinant proteins in various applications, such as Western blotting, immunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
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Lab products found in correlation
Champ2, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor mixture, by Merck Group (1 mentions)
Sheep anti mouse igg paramagnetic beads, by Thermo Fisher Scientific (1 mentions)
Luminescent substrate, by Merck Group (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
Hybond ecl nitrocellulose membrane, by GE Healthcare (1 mentions)
Qubit protein assay kit, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Ultrapure dnase rnase free distilled water, by Thermo Fisher Scientific (1 mentions)
Zeocin antibiotic, by Thermo Fisher Scientific (1 mentions)
Live dead fungalight yeast viability kit, by Thermo Fisher Scientific (1 mentions)
Microseal b adhesive sealing film, by Bio-Rad (1 mentions)
Yeastar genomic dna isolation kit, by Zymo Research (1 mentions)
6 protocols using anti his mab
1
Immunoprecipitation of FT2Fc from Cell Cultures
2
Quantitative Western Blot Analysis
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Cells were lysed in buffer containing 50 mM HEPES, 150 mM KCl, 2 mM EDTA, 0.5% NP-40, and a protease inhibitor cocktail. Lysates (40 μg) were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membranes were probed with anti-spike protein mAb (1A9) (GeneTex), anti-p24 mAb (AG3.0), anti-His mAb (Invitrogen), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mAb (Life Technologies) followed by goat anti-mouse horseradish peroxidase (HRP)-conjugated second antibody (Sigma). The membrane was treated with a luminescent substrate (Millipore), and the band intensities were quantified on an iBright CL1000 imager.
3
DR1 Binding Assay Protocol
4
DR1 Binding Assay Protocol
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Samples were prepared by incubating DR1 (1μM) with various combinations of the following: rHA1 (2.7μM), HA(306–318) (300μM), and DM (1μM) in citrate phosphate buffer pH 5.0/0.05% NaN3 for 3h in 37°C waterbath. Following incubation, pH was adjusted to 7.4 with either 0.2M sodium phosphate dibasic or 1M Tris-HCl pH 8.0 and the samples were mixed with a modified Laemmli buffer with 0.1% SDS final concentration and no reducing agents. The samples were resolved without boiling on 12% polyacrylamide gels and were silver stained63 (link). For Western Blot experiments, various combinations of DR1(1μM), DM (0.5μM), rHA1 (2μM), or HA(306–318) (300μM) were incubated in citrate-phosphate buffer pH 5.0 overnight at 37°C. The pH was readjusted to 7.4 and the samples were mixed with Laemmli buffer containing 0.1% SDS and 2.5% 2ME. They were either not boiled (room temperature), or boiled prior to loading on 10% SDS-PAGE. Samples on gel were transferred to PVDF membranes; DR1/Protein complexes were detected by anti-His mAb (1:10,000) (Invitrogen) or CHAMP2 (1:4,000) (Rabbit polyclonal anti DR1 serum (Gift from Dr. Larry Stern), followed by staining with anti-mouse HRP (1:5,000) or anti-Rabbit HRP Ab (1:5,000) respectively.
5
Quantification and Visualization of Proteins
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Total protein was quantified using the Qubit Protein Assay Kit (Life Technologies – Invitrogen, # Q33211) in a Qubit fluorometer (Life Technologies – Invitrogen). For SDS-PAGE separation, protein fractions were mixed with 5x reducing sample buffer (Thermo Scientific, #39000), resolved on 12% (w/v) polyacrylamide gels, and visualized by staining with Coomassie blue G250 [21 (link)]. For immunoblot analysis, proteins were electroblotted onto Hybond ECL nitrocellulose membrane (GE Healthcare Life Sciences, #RPN2020D). The membrane was washed in Tris-buffered saline (TBS) for 5 min, blocked with 5% nonfat milk in TTBS (TBS with 0.1% Tween 20) for 1h by shaking at room temperature, and probed with either primary anti-Nef MAb (Thermo Scientific, #MA1-71507) or anti-His MAb (Thermo Scientific, #MA1-213157) with shaking at 4°C overnight. After washing with TTBS, membrane was incubated with secondary HRP-conjugated IgG (H+L) antibody (Thermo Scientific, #32430) and protein bands were detected using SuperSignal West Pico Chemiluminescent substrate (Thermo Scientific, #34080). Images were captured using FluorChem M Imager (Protein Simple).
6
Expression and Purification of Dengue Virus Antigen
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All the molecular biology enzymes, P. pastoris codon optimized EDIII gene of dengue virus serotype-1 (DENV-1) with 6×-His tag at 3′end, zeocin antibiotic, LIVE/DEAD FungaLight yeast viability kit, 96-DWP (square wells with V-shaped bottom; 2.2 ml total volume), breathable rayon tape, anti-His mAb and UltraPure DNase/RNase-free distilled water were purchased from Thermo Fisher Scientific Corporation, USA. 96-DWP (square wells with U-shaped bottom; 2.2 ml total volume per well) was from Genetix Biotech Asia Pvt Ltd, India. Goat anti-mouse IgG-H&L-chain was procured from Jackson ImmunoResearch Laboratories, Inc. USA. N1-europium chelate was synthesized at University of Turku, Finland. YeaStar genomic DNA isolation kit was purchased from Zymo Research, CA, USA. 2x SSO EvoGreen mix, hard-shell white 96-well PCR plate with clear wells and microseal ‘B’ adhesive sealing film were obtained from Bio-Rad Laboratories, CA, USA. All the other chemicals were procured from Sigma-Aldrich Corporation, USA and culture media and casamino acids were purchased from Becton, Dickinson and Company, USA. The casamino acids (CA) is acid hydrolysed casein with low sodium chloride and iron concentrations (BactoTM casamino acids Cat # 223050). Peptone is an enzymatic digest of animal protein (BactoTM peptone Cat # 211677). Primers for quantitative PCR (qPCR), were synthesized by IDT, Singapore.
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