We prepared lipid vesicles by drying dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), or a 1:1 (w/w) mixture of DOPG and DOPC under constant argon flow to avoid oxidation. We re-suspended the resultant lipid film in buffer (100 mM NaCl, 20 mM Tris [pH 7.5]) and sonicated the mixture for 15 min to generate vesicles. Final lipid concentrations were 2 mg/ml. We mixed 100 μl of vesicle suspension with equal amounts of wild-type or mutant EphA2 FN domain 2 protein, incubated these at room temperature for 10 min, and centrifuged them at >20,000 × g for 10 min. We repeated the experiments seven times. For each set the bound protein fractions were visualized by western blot using mouse anti-pentaHis antibody (Qiagen). Data analysis was done with ImageJ (Schneider et al., 2012 (link)). In each set, we normalized the data using the corresponding value measured for wild-type protein pelleted with DOPC vesicles. After normalization, we calculated averages and SEM from all experiments.
Mouse anti penta his antibody
Manufactured by Qiagen
Sourced in Germany
The Mouse anti-penta-His antibody is a laboratory reagent used for the detection and purification of recombinant proteins containing a His-tag. It specifically binds to the His-tag sequence, allowing for the identification and isolation of target proteins in various applications.
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Lab products found in correlation
Mini protean tgx stain free protein gel, by Bio-Rad (2 mentions)
Irdye 800cw, by LI COR (2 mentions)
Irdye 680rd, by LI COR (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Trans blot turbo transfer system, by Bio-Rad (2 mentions)
Immuno blot pvdf membrane, by Bio-Rad (1 mentions)
Coomassie brilliant blue r 250, by Merck Group (1 mentions)
Odyssey system, by LI COR (1 mentions)
Odyssey software, by LI COR (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Semi dry blotting system, by ATTO Corporation (1 mentions)
Iblot, by Thermo Fisher Scientific (1 mentions)
Odyssey clx imaging system, by LI COR (1 mentions)
Irdye 800cw donkey anti mouse igg secondary antibody, by LI COR (1 mentions)
Iblot 2 transfer stack, by Thermo Fisher Scientific (1 mentions)
Mini protean tgx stain free precast gel, by Bio-Rad (1 mentions)
8 protocols using mouse anti penta his antibody
1
Lipid Vesicle Protein Binding Assay
2
Protein Visualization and Western Blot Analysis
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Purified protein fractions were separated on a Mini-PROTEAN TGX Stain-Free protein gel (Bio-Rad Laboratories, Inc.). Separated protein bands were visualized using “Stain Free Gel” application mode on ChemiDoc MP Imaging System (Bio-Rad Laboratories, Inc.). Protein gel was transferred to a nitrocellulose membrane (Bio-Rad, cat. #1704271) using a Trans-Blot Turbo Transfer System (Bio-Rad Laboratories, Inc.). Membranes were blocked in PBST containing 5% milk for 30 min at 22 °C. The membranes were then incubated with primary antibodies for GFP (custom anti-GFP rabbit polyclonal (provided by Foley lab, Memorial Sloan Kettering Cancer Center) at a dilution of 1:5,000) and His (mouse anti-penta-His antibody (Qiagen, cat. #34660) at a dilution of 1:10,000) in PBST + 5% milk overnight at 4 °C. The membranes were washed three times with PBST and were incubated with goat anti-rabbit IgG polyclonal antibody (IRDye 800CW (LI-COR Biosciences cat. #925-32211) at dilution of 1:10,000) and goat anti-mouse IgG polyclonal antibody (IRDye 680RD, LI-COR Biosciences #926-68070 at a dilution of 1:10,000) as the secondary antibodies in PBST + 5% milk for 1 hr at 22° C. The membranes were washed three times with PBST and imaged using a LI-COR (LI-COR Biosciences) and analyzed by ImageJ75 .
3
Protein Detection and Quantification Protocol
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SDS–polyacrylamide gel electrophoresis was performed using standard procedures. Proteins were stained with Coomassie brilliant blue R250 (Sigma-Aldrich Chemie GmbH, Munich, Germany) or transferred to Immuno-Blot PVDF membrane (Bio-Rad, Hercules, CA, USA). Recombinant immunotoxins were detected with mouse anti-penta-His antibody (Qiagen). Cleavage of poly(ADP-ribose) polymerase (PARP) was analyzed using whole-cell protein extracts prepared from 1 × 106 cells as previously published.37 (link) Full-length PARP and its specific cleaved product were detected using mouse anti-human PARP antibody (Cell Signaling Technology, Danvers, MA, USA). Horseradish peroxidase-conjugated goat anti-mouse antibodies (Dianova, Hamburg, Germany) were used as secondary antibodies. Detection of bound antibody was performed with SuperSignal West Dura Extended Duration Substrate (Pierce, Rockford, IL, USA).
4
Double Cysteine Mutant Analysis of MCU
5
Immunoblotting of Plasmodium vivax Proteins
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The PvPHIST/CVC-8195-NT and CT proteins were separated by SDS-PAGE under reducing conditions. Then, proteins were transferred from the SDS-PAGE gel to PVDF membranes (0.45 μm, Millipore, Billerica, Massachusetts, USA) by using a semi-dry blotting system (ATTO Corp., Tokyo, Japan) in a semi-dry transfer buffer (50 mM Tris, 190 mM glycine, 3.5 mM SDS, 20% methanol) at a constant 400 mA for 40 min and followed by blocking with 5% skim milk in PBS containing 0.2% Tween 20 (PBS-T) overnight. The mouse anti-penta His antibody (Qiagen, Hilden, Germany) and P. vivax or healthy control sera (1:200) were diluted into PBS/T and used as the first antibody. His-tagged recombinant proteins were finally detected by using goat anti-mouse (1:10,000, PBS/T) or goat anti-human (1:20,000, PBS/T) antibodies conjugated IRDye® (LI-COR Bioscience, Lincoln, Nebraska, USA). Reacted membrane was scanned by Odyssey infrared imaging system (LI-COR Biosciences) and results were analyzed by Odyssey software (LI-COR Biosciences).
6
HDL Proteome Analysis of PLY and NanA
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Hirudin anticoagulated blood was incubated in the presence or absence of 200 nM PLY or 200 nM PLY and 1.2 μM NanA for 1 h at 37 °C in a rotator. Plasma was separated from blood by centrifugation (10 min 1,000 × g) and HDL samples were prepared by sequential flotation ultracentrifugation using potassium-bromide for density adjustment as described above. The HDL proteomes were analyzed by MS/MS and western blotting (WB). For WB 40 μl of the samples and 50 ng of PLY were run on SDS-PAGE using Mini-PROTEAN TGX Stain-Free precast gels (Bio-Rad) under non-reducing conditions for 75 min at 150 V. The proteins were transferred (iBlot, Thermo Fisher Scientific) to nitrocellulose membrane (iBlot™ 2 Transfer Stack, Invitrogen) and blocked with 3% milk/PBS. The membrane was then incubated with 0.4 μg/ml of mouse anti-penta-His antibody (Qiagen, Hilden, Germany) in 0.3% milk/PBS for 1 h at 37 °C and after washing with PBS with 1:5000 dilution of IRDye® 800CW Donkey anti-mouse IgG Secondary Antibody (LI-COR) for 1 h at 37 °C in 0.3% milk-PBS. The membrane was washed with PBS and imaged using Odyssey® CLx Imaging System (LI-COR).
7
Binding Activity of B27 mAb to IFN-γ
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To test the binding activity of B27 mAb to IFN-γ, the plates were coated with B27 mAb (5 μg/mL, 50 μL per well) in bicarbonate buffer overnight at 4 °C. Mouse anti-Penta-His antibody (Qiagen, Germany) was coated into separating wells for indicating the presence of IFN-γ in the soluble fractions. After washing, non-specific protein binding was blocked with 2% skimmed milk in PBS. Bacterial soluble fractions containing IFN-γ WT, T27A, T27AF29AL30A, or T27-L33 deletion (dilution 1:10, 50 μL per well) were added to wells and incubated for 1 h at room temperature. The wells were washed four times with 0.05% Tween 20 in PBS before the addition of horseradish peroxidase (HRP)-conjugated anti-6 × His mAb (BioLegend, San Diego, CA). After 1 h incubation, plates were washed four times, and TMB substrate was subsequently added. The reaction was stopped by adding 1 N HCl, and absorbance at 450 nm was determined using an ELISA microplate reader.
8
Western Blot Analysis of Protein Samples
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Purified protein fractions were separated on a Mini-PROTEAN TGX Stain-Free protein gel (Bio-Rad Laboratories, Inc.). Separated protein bands were visualized using "Stain Free Gel" application mode on ChemiDoc MP Imaging System (Bio-Rad Laboratories, Inc.). Protein gel was transferred to a nitrocellulose membrane (Bio-Rad, cat. #1704271) using a Trans-Blot Turbo Transfer System (Bio-Rad Laboratories, Inc.). Membranes were blocked in PBST containing 5% milk for 30 min at 22 °C. The membranes were then incubated with primary antibodies for GFP (custom anti-GFP rabbit polyclonal (provided by Foley lab, Memorial Sloan Kettering Cancer Center) at a dilution of 1:5,000) and His (mouse anti-penta-His antibody (Qiagen, cat. #34660) at a dilution of 1:10,000) in PBST + 5% milk overnight at 4 °C. The membranes were washed three times with PBST and were incubated with goat anti-rabbit IgG polyclonal antibody (IRDye 800CW (LI-COR Biosciences cat. #925-32211) at dilution of 1:10,000) and goat anti-mouse IgG polyclonal antibody (IRDye 680RD, LI-COR Biosciences #926-68070 at a dilution of 1:10,000) as the secondary antibodies in PBST + 5% milk for 1 hr at 22° C. The membranes were washed three times with PBST and imaged using a LI-COR (LI-COR Biosciences) and analyzed by ImageJ 75 .
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