SDS–PAGE was performed with 13.5% acrylamide gels in denaturing conditions. For the western blots, protein samples were mixed with 4 × SDS loading buffer (1 M Tris–HCl, pH 6.8, 8% SDS, 40% glycerol, 0.4 M dithiothreitol, 0.8% bromophenol blue) and boiled for 10 min at 98 °C before separation by 13.5% SDS–PAGE. The proteins were transferred onto nitrocellulose membranes (Whatman, Kent, UK). After the membranes were blocked with 3% nonfat milk at room temperature for 30 min, they were probed with an anti-His tag monoclonal antibody (Invitrogen, Carlsbad, CA, USA), followed by alkaline phosphatase-conjugated AffiniPure goat anti-mouse IgG (Beijing Dingguo, Inc., Beijing, China). Subsequently, the immunoreaction was detected with 5-bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium chloride solutions.
Anti his tag monoclonal antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The anti-His tag monoclonal antibody is a laboratory reagent used to detect and purify recombinant proteins containing a histidine (His) tag. It binds specifically to the His tag, allowing for the identification and isolation of the target protein from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Cytiva (3 mentions)
Amicon ultra 15, by Merck Group (1 mentions)
Ecl plus substrate, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Protein marker, by Thermo Fisher Scientific (1 mentions)
Protein marker, by Bio-Rad (1 mentions)
Recombinant plasmids, by GenScript (1 mentions)
Bl21 de3 competent cells, by Transgene (1 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Tiangen Biotech (1 mentions)
H 7650, by Hitachi (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Horseradish peroxidase conjugated anti mouse igg antibody, by Merck Group (1 mentions)
Beyoecl moon kit, by Beyotime (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Desalting column, by Thermo Fisher Scientific (1 mentions)
Nunc cell factory system, by Thermo Fisher Scientific (1 mentions)
Histrap column, by GE Healthcare (1 mentions)
Anti β actin peroxidase antibody, by Merck Group (1 mentions)
Anti rabbit igg hrp, by Cell Signaling Technology (1 mentions)
Anti mouse igg hrp, by Cell Signaling Technology (1 mentions)
10 protocols using anti his tag monoclonal antibody
1
SDS-PAGE and Western Blotting Protocol
2
Purification and Characterization of Recombinant Proteins
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The sample solution containing the target protein was obtained by centrifugation, ultrasonic fragmentation and centrifugation. The target proteins were purified by affinity chromatography using Ni Focurose 6FF (IMAC) (HUIYAN Bio, China). After overnight dialysis, the purified protein was filtered and concentrated by 100 kDa Amicon Ultra-15 centrifugal filters (Merck Millipore Ltd). The final concentration of the protein was determined by a BCA protein assay kit (Beyotime, China). Finally, four target proteins denoted ferritin, YaH3F, YaH4F, and YaH5F were obtained and stored at − 80 °C.
The expression of all proteins was verified by using reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The purified protein was used to verify the effect of ferritin self-assembly through 6% nonreducing SDS–PAGE, followed by blotting and then analysis by Western blotting using an anti-His tag monoclonal antibody (Invitrogen, Carlsbad, CA, USA). Western blotting was performed by using the ECL Plus substrate (Beyotime Biotech, Inc., China).
The expression of all proteins was verified by using reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The purified protein was used to verify the effect of ferritin self-assembly through 6% nonreducing SDS–PAGE, followed by blotting and then analysis by Western blotting using an anti-His tag monoclonal antibody (Invitrogen, Carlsbad, CA, USA). Western blotting was performed by using the ECL Plus substrate (Beyotime Biotech, Inc., China).
3
Amyloid-beta Peptide Production and Detection
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Aβ42 was synthesized by GL Biochem (Shanghai, China). Recombinant plasmids were synthesized by GenScript (Nanjing, China). BL21(DE3) competent cells were purchased from TransGen Biotech (Beijing, China). 3,3,5,5′-tetramethylbenzidine (TMB) was purchased from Tiangen Biotech (Beijing, China). Nitrocellulose membrane was purchased from Whatman (Maidstone, UK). Non-pre-stained Protein Marker was purchased from Thermo Fisher (Rockford, USA). Horseradish peroxidase-labeled (HRP-labeled) goat anti-human IgG (H + L) was purchased from Sino Biological (Beijing, Chian). Anti-His-tag monoclonal antibody was purchased from Invitrogen (Carlsbad, CA, USA). Protein Marker was purchased from Bio-Rad (CA, USA). Mouse monoclonal antibody, β-Amyloid (B-4), recognizing Aβ42 was purchased from Santa Cruz Biotechnology, INC.
4
SDS-PAGE Analysis of ScFv Purity
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ScFv purity was assessed using SDS-PAGE. The samples were boiled in a SDS sample-loading buffer containing dithiothreitol. The ScFvs were electrophoresed on a 12% SDS-PAGE gel, followed by blotting, and then analysis was performed using Western blotting with an anti-His-tag monoclonal antibody (Invitrogen, Carlsbad, CA, USA). Western blotting was visualized using a ECL Plus substrate (Tanon Science and Technology, Shanghai, China).
5
Native Protein Structure Analysis
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The molecular weights (MW) of the proteins were estimated by 13.5% SDS-PAGE. Native-PAGE was used to identify the 24-mer form of the protein: the native-PAGE gels did not contain 10% SDS and the samples were not treated with β-mercaptoethanol prior to separation.
For Western blots, the proteins were transferred onto nitrocellulose membranes (Whatman, Kent, UK) after separated by 13.5% SDS-PAGE. An anti-His tag monoclonal antibody (Invitrogen, USA) was used to analysis the particles.
The morphological characteristics of the proteins were observed using a TEM (H-7650, Hitachi, Japan). Observations were conducted by TEM (H-7650, Hitachi, Japan) with an accelerating voltage of 80 kV, and images (50 k magnification) were obtained with a CCD camera system.
For Western blots, the proteins were transferred onto nitrocellulose membranes (Whatman, Kent, UK) after separated by 13.5% SDS-PAGE. An anti-His tag monoclonal antibody (Invitrogen, USA) was used to analysis the particles.
The morphological characteristics of the proteins were observed using a TEM (H-7650, Hitachi, Japan). Observations were conducted by TEM (H-7650, Hitachi, Japan) with an accelerating voltage of 80 kV, and images (50 k magnification) were obtained with a CCD camera system.
6
Recombinant Protein E2e Characterization
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The expression of a recombinant protein named E2e was evaluated by SDS-PAGE and Western blot. Thus, the purified protein was diluted in sample loading buffer for SDS-PAGE and boiled at 100°C for 5 min. The recombinant protein was visualized in SDS-PAGE stained with Coomassie brilliant blue. Moreover, the recombinant protein was transferred onto a nitrocellulose membrane (Millipore Darmstadt, Germany) and probed with an anti-His tag monoclonal antibody (Invitrogen, Carlsbad, CA, USA), an anti-myc monoclonal antibody (Invitrogen, Carlsbad, CA, USA), or an anti-BVDV-E2 mouse monoclonal antibody (VMRD, Pullman, WA, USA), followed by a secondary horseradish peroxidase-conjugated anti-mouse IgG antibody (Sigma Aldrich, Saint Louis, MO, USA), and visualized by chemiluminescence (Thermo Fisher Scientific, Waltham, MA, USA).
7
Validating rAAV8 Transduction Efficiency
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After purification of rAAV8, 100 μL Cap-rAAV, RFP-rAAV, and Cap-pSec was added to each well of a 6-well plate to find out whether the purified rAAV8 could infect HEK293T cells. By 72 h post-infection, the supernatant of each well was collected. The expressed protein in HEK293T cells was analyzed under reducing conditions by western blot. The expressed proteins were separated on 12.0% polyacrylamide gels and transferred onto nitrocellulose membranes (Bio-Rad, USA), which were blocked with 3% skim milk in PBS at room temperature for 1.5 h and probed with an anti-His tag monoclonal antibody (Invitrogen, USA). Subsequently, the membranes were probed with a horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Beijing Dingguo Inc., China), and then the immunoreaction was detected using a BeyoECL Moon kit (Beyotime Biotechnology, China). The fluorescence of RFP expressed in HEK293T cells was observed using a fluorescent microscope.
8
Recombinant C4BP-ANG1 and ANG1-C4BP Production
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Production of recombinant C4BP-ANG1 and ANG1-C4BP was performed in HEK293 clonal expression system that was described previously (35) . Briefly, the constructs in expression vector pcDNA3 were transfected to HEK293 cells, and selected in DMEM medium supplemented with 800 µg/ml of G418 until individual colonies were visible under microscope. Individual colonies were then selected and expanded in separate wells. The expression of the recombinant protein was measured by ELISA using anti-His tag monoclonal antibody (Invitrogen) to capture and biotinylated anti-ANG1 antibody for detection (R&D System). The clones showing the highest titers of the recombinant protein were selected. Protein production was in a Nunc CellFactory system (ThermoFisher) using serum-free culture. The medium was centrifuged to clear the cell debris, followed by concentrating and buffer exchange into IMAC binding buffer using Vivaflow 100 system (Sartorius). The resulting protein solution was purified by Histrap column (GE Healthcare) and then buffer exchanged into DPBS by using desalting column (Thermo). For further scale-up production of the C4BP-ANG1 variants, an alternative CHO expression system was used, as described previously (36, 37) .
9
Integrin Binding Assay Protocol
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Integrin binding assays were performed as described (Nishiuchi et al., 2006 (link)). Microtiter plates were coated with 10 nM recombinant human Osteolectin, recombinant human Pro-Collagen 1α, or Bovine Serum Albumin (BSA, Sigma A3156) overnight at 4°C, and then blocked with 10 mg/ml BSA. 6xHis tagged recombinant human integrin heterodimers were purchased from R and D Systems. The plates were incubated with integrins in TBS buffer (50 mM Tris-Cl, pH 7.5 150 mM NaCl) with 1 mM MnCl2, then washed with TBS containing 1 mM MnCl2, 0.1% BSA, and 0.02% Tween 20, followed by quantification of bound integrins by an enzyme-linked immunosorbent assay using an anti-His tag monoclonal antibody (Thermo Fisher Scientific, clone 4E3D10H2/E3) followed by a horseradish peroxidase-conjugated anti-mouse secondary antibody. After washing, bound HRP was detected using SureBlue TMB Microwell Peroxidase Substrate (KPL) and the reaction was stopped with TMB stop solution (KPL). The optical density was measured at 450 nm.
10
Western Blot Analysis of Midgut Proteins
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Midguts were homogenized in 2x Laemmli sample buffer (Bio-Rad), boiled for 5 min, and centrifuged at 10,000 g for 10 min. The supernatants were separated by SDS-PAGE and transferred to a nitrocellulose membrane. After blocking with 5% non-fat dried milk in a Tris-buffered saline buffer (20 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 0.1% Tween 20, pH 7.5) (TBST) for 1 h, the membrane was incubated in the blocking solution overnight at 4°C with anti-His tag monoclonal antibody (Thermo Scientific) or anti-human collagen IV polyclonal antibodies (Abcam, Cambridge, MA, USA). Following overnight incubation with the primary antibodies, membranes were washed three times (6 min/wash) in TBST. Membranes were incubated with anti-rabbit IgG-HRP or anti-mouse IgG-HRP (Cell Signaling Technology, Danvers, MA, USA) at RT for 2 h and then treated for 1 min with SuperSignal West Pico chemiluminescent substrate (Pierce, Waltham, MA, USA). The immunoreactive proteins were visualized by exposing the membrane to an x-ray film. Anti-β-actin-peroxidase antibody (Sigma) was used to detect the loading control in each lane.
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