SPC-A1, PC9 and H69 cells were incubated in medium containing 10 μg scFv or scFv-9R at 37°C for 6 h, followed by an incubation with the anti-His mouse monoclonal antibody (#34660, Qiagen, CA, USA) at room temperature for 2 h. The cells were then stained with FITC-labeled goat anti-mouse secondary antibody (Boster, Wuhan, China) at 4°C for 1 h. After washing with ice-cold PBS, the fluorescence intensity of cells was recorded using a FACSCalibur flow cytometer (BD Bioscience, Mountain View, CA, USA). Data from 10,000 cells were collected and analyzed using the Cell-Quest software (BD Bioscience). To examine the targeted delivery of siRNA, 10 μg scFv, scFv-9R or BSA were pre-mixed with 20 nM FAMsi at room temperature for 30 min and then added into the culture medium of SPC-A1, PC9 and H69 cells. Six hours later, the cells were harvested and washed with cold PBS extensively and then analyzed by FCM. Transfection of FAMsi using Lipofectamine served as a positive control.
Mouse anti his monoclonal antibody
Manufactured by Qiagen
Sourced in United States
The Mouse anti-His monoclonal antibody is a laboratory reagent that specifically recognizes and binds to the histidine (His) tag, a commonly used protein tag for purification and detection purposes. This antibody can be used to detect and localize His-tagged recombinant proteins in various applications, such as Western blotting, immunoprecipitation, and immunohistochemistry.
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Clarity western ecl substrate, by Bio-Rad (1 mentions)
8 protocols using mouse anti his monoclonal antibody
1
Targeted Delivery of scFv-Mediated siRNA
2
Immunoblotting for Protein Detection
3
Molecular Construction and Characterization of EV71 IRES
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To make full-length and truncated PTB expression constructs, cDNA from T98G cells was used as template for amplification by specific primers (Supplementary Table S1). The resultant PCR products were cloned into pcDNA4.0 vector (Invitrogen, USA). The secondary structure of EV71 5′UTR was predicted by the mfold web server and used to design full-length and truncated IRES probes (Bellaousov et al. 2013 (link)), which were then amplified from EV71 cDNA using specific primers (Supplementary Table S1). The EV71 IRES element contains five major stem-loops (SL II–VI). The PCR products were cloned into pGEM-3zf vector (Promega, USA) to generate pGEM-3zf-IRES, pGEM-3zf-SL II (121–180), SL III (190–230), SL IV (241–450), SL V (451–563), SLVI (564–742, stem-loop VI, and linker region), respectively. All the constructs were confirmed by DNA sequencing.
The siRNAs against PTB and negative control siRNA (siNC) were purchased from Invitrogen (Cat. AM16708). Antibodies used in this study were as follows: rabbit anti-PTB polyclonal (generated in our lab) antibody, mouse anti-β-actin monoclonal antibody (1:3000, Sigma, USA), mouse anti-CREB monoclonal antibody (1:1000, CST, USA), mouse anti-his monoclonal antibody (1:1000, Qiagen, Germany), rabbit anti-EV71 VP1 polyclonal antibody (1:1000, Abnova, Taiwan, China), and mouse anti-EV713C monoclonal antibody (1:200, Millipore, USA).
The siRNAs against PTB and negative control siRNA (siNC) were purchased from Invitrogen (Cat. AM16708). Antibodies used in this study were as follows: rabbit anti-PTB polyclonal (generated in our lab) antibody, mouse anti-β-actin monoclonal antibody (1:3000, Sigma, USA), mouse anti-CREB monoclonal antibody (1:1000, CST, USA), mouse anti-his monoclonal antibody (1:1000, Qiagen, Germany), rabbit anti-EV71 VP1 polyclonal antibody (1:1000, Abnova, Taiwan, China), and mouse anti-EV713C monoclonal antibody (1:200, Millipore, USA).
4
Ail Protein Binding Assay
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Binding assays were performed as described (28 (link), 40 (link)). Briefly, 96-well plates (Nunc) were preadsorbed with Vn-HX (5 μg/ml), Vn (5 μg/ml; Sigma), or gelatin (20 μg/ml; Sigma), then blocked with tris-buffered saline (TBS) containing 3% milk, and washed with TBS supplemented with 0.05% Tween 20. Recombinant C-terminal His-tagged Ail (Ail-His) was purified, refolded, and concentrated to 80 μg/ml in buffer with 4 mM decylphosphocholine and then added in decreasing concentrations to the preadsorbed wells. After incubating overnight at 4°C, bound Ail-His was detected with mouse anti-His monoclonal antibody (Qiagen), secondary goat anti-mouse antibody conjugated to horseradish peroxidase (Sigma), and the horseradish peroxidase substrate o-phenylenediamine (Pierce).
5
Western Blotting and Dot Blot Analysis
6
Protein Characterization via Chromatography and Spectroscopy
7
SUMO-VP6 Fusion Protein Purification and Validation
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Recombinant SUMO-VP6 fusion protein purified from the Ni2+-NTA column was electrophoresed on a 10% SDS-PAGE gel. The purity of the column-purified recombinant SUMO-VP6 and the specificity of the polyclonal antiserum were determined by immunoblot analysis of the protein.
The SDS-PAGE gel was electrotransferred to nitrocellulose membranes for 1 hour at 100 mV using a Bio-Rad Trans-Blot® apparatus. The nitrocellulose membranes were incubated with blocking solution (1× PBS, 5% fat-free milk powder, and 0.3% Tween 20) for 1 hour at room temperature and then incubated with either the anti-VP6 antiserum (1:2,500 dilution) or the mouse anti-His monoclonal antibody (Qiagen) in blocking solution for a further 2 hours. Preimmune serum collected from the same mouse was used as a control (data not shown). The membrane was washed three times with blocking solution and incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin (Ig)-G antibody (Sigma-Aldrich, St Louis, MO, USA) for 1 hour. After the membrane had been washed three times with blocking solution and twice with Tris-buffered saline (50 mM Tris HCl, 150 mM NaCl, pH 7.5), antibodies were detected by adding to the membrane SuperSignal™ West Dura extended-duration substrate (Thermo Fisher Scientific). Results of all Western blot experiments were analyzed using a Bio-Rad ChemiDoc™ imaging system.
The SDS-PAGE gel was electrotransferred to nitrocellulose membranes for 1 hour at 100 mV using a Bio-Rad Trans-Blot® apparatus. The nitrocellulose membranes were incubated with blocking solution (1× PBS, 5% fat-free milk powder, and 0.3% Tween 20) for 1 hour at room temperature and then incubated with either the anti-VP6 antiserum (1:2,500 dilution) or the mouse anti-His monoclonal antibody (Qiagen) in blocking solution for a further 2 hours. Preimmune serum collected from the same mouse was used as a control (data not shown). The membrane was washed three times with blocking solution and incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin (Ig)-G antibody (Sigma-Aldrich, St Louis, MO, USA) for 1 hour. After the membrane had been washed three times with blocking solution and twice with Tris-buffered saline (50 mM Tris HCl, 150 mM NaCl, pH 7.5), antibodies were detected by adding to the membrane SuperSignal™ West Dura extended-duration substrate (Thermo Fisher Scientific). Results of all Western blot experiments were analyzed using a Bio-Rad ChemiDoc™ imaging system.
8
Detecting Recombinant E2 Expression in Sf9 Cells
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For detection of expressed E2, Sf9 cells were infected with recombinant baculovirus Bac-CMV-E2-gp64 or Bac-WT as a negative control. Three days post infection, cells supernatants (5 µl) were mixed with 2 × SDS-PAGE sample buffer, boiled and loaded to a 10% SDS-PAGE based on standard protocols [34 ]. For western blot analysis, 20 μl of supernatants were loaded on SDS-PAGE and the proteins were transferred to polyvinylidene difluoride (PVDF) membranes. Then, it was incubated with a mouse anti-His monoclonal antibody (1:15,000; QIAGEN, Valencia, CA) as the primary antibody. The secondary antibody was horseradish peroxidase (HRP) conjugated goat anti-mouse IgG antibody (1:3000; Southern Biotech, Birmingham, AL). Reactive bands were visualized using an enhanced chemiluminescence (ECL) Kit (Clarity™ Western ECL Substrates, Bio Rad) and light emission was detected by exposing the membrane to a Hyper film ECL (GE-Amersham).
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