Protein integrity and purity was assessed by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western blotting using an anti-His monoclonal antibody (Santa Cruz Biotechnology). The molecular mass of T22-HA2-GFP-H6 and T22-GFP-HA2-H6 proteins was determined by Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) mass spectrometry, while the volume size distribution was determined by Dynamic Light Scattering (DLS) at 633 nm in a Zetasizer Nano ZS (Malvern Instruments Limited). Additionally, fluorescence of the GFP-based nanoparticles was determined by a Varian Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies), through an excitation wavelength at 450 nm and detection at 510 nm. Protein stability, was assessed in human sera (Sigma-Aldrich) at 37 ºC for 0, 2, 5 and 24 h. Then, protein integrity was evaluated by Western blot using an anti-His monoclonal antibody (Santa Cruz Biotechnology) for the specific immunodetection of the His-tag. Additionally, Precision Plus Protein All Blue Standards (BioRad) was used to confirm the expected molecular weight of the samples.
Anti his monoclonal antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Anti-His monoclonal antibody is a laboratory reagent used to detect and purify proteins containing a histidine tag. It binds specifically to the histidine tag, which is a common affinity tag used to facilitate protein purification and detection.
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Lab products found in correlation
Ecl plus kit, by GE Healthcare (1 mentions)
Nitrocellulose, by Merck Group (1 mentions)
Hrp conjugated goat anti mouse igg, by Santa Cruz Biotechnology (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Tgx stain free fastcast, by Bio-Rad (1 mentions)
Zetasizer nano z, by Malvern Panalytical (1 mentions)
Cary eclipse, by Agilent Technologies (1 mentions)
Histrap excel column, by GE Healthcare (1 mentions)
Kta pure system, by GE Healthcare (1 mentions)
Histrap hp column, by GE Healthcare (1 mentions)
Human sera, by Merck Group (1 mentions)
Varian cary eclipse fluorescence spectrophotometer, by Agilent Technologies (1 mentions)
Precision plus protein all blue standard, by Bio-Rad (1 mentions)
Tgx stain free gel, by Bio-Rad (1 mentions)
7 protocols using anti his monoclonal antibody
1
Comprehensive Protein Characterization Methodology
2
Evaluation of AQ Binding to TseF
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The binding capacity of TseF to different 2-alkyl-4(1H)-quinolone (AQ) molecules were evaluated by fat western blotting as reported67 . Briefly, AQs were solubilized in chloroform as a stock solution of 10 mM, and a minimum of 10 μl containing 0.1, 0,2 or 0.5 μM of AQs were spotted onto nitrocellulose (NitroBind, MSI, USA). The dotted membrane was dried at 24 °C for 1 h and the nitrocellulose was then incubated with 3% (w/v) fatty acid-free BSA (A-6003, isolated by cold ethanol precipitation; Sigma) in TBST (10 mM Tris (pH 8.0), 140 mM NaCl and 0.1% (v/v) Tween 20) for 1 h and then placed in a solution containing the His-tagged proteins (TseF or Fur) diluted in TBST (0.5 mg ml−1) at 4 °C overnight with shaking. The membrane was washed with TBST three times for 10 min each and then incubated with anti-His monoclonal antibody (Santa Cruz Biotechnology, USA) diluted at 1:200 in TBST for 1 h at 24 °C. The incubated membrane was then washed three times for 10 min in TBST at 24 °C and then incubated with 1:10,000 dilution of horseradish peroxidase-conjugated secondary antibodies (Shanghai Genomics) for 1 h. Signals were detected using the ECL Plus Kit (GE Healthcare, Piscataway, NJ) following the manufacturer's specified protocol.
3
Western Blot Analysis of Recombinant Proteins
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Purified N-terminal
histidine-tagged full-length and truncated E2 fusion proteins were
separated on a 10% SDS-PAGE gel along with protein marker, positive
control (histidine-tagged fusion protein), and negative control (bovine
serum albumin, BSA) proteins and transferred to a nitrocellulose membrane
(Bio-Rad Laboratories). The membrane was blocked using 3% (w/v) BSA
in Tris-buffered saline (TBS) for 1 h at room temperature. Subsequently,
the membrane was washed four times with 1× TBS containing 0.05%
Tween-20 (TBST). Next, the membrane was incubated with the anti-His
monoclonal antibody (Santa Cruz Biotechnology) at 1:1000 dilution
for 1 h at room temperature. After washing four times with TBST, the
membrane was incubated for 1 h at room temperature with HRP-conjugated
goat antimouse IgG (Santa Cruz Biotechnology) at 1:5000 dilution.
After washing four times with TBST and once with TBS, the membrane
was incubated with a 3,3′-diaminobenzidine (DAB) substrate
solution (0.5 mg/mL) at room temperature. Color development was stopped
by washing with water, following which the blots were air-dried and
stored.
histidine-tagged full-length and truncated E2 fusion proteins were
separated on a 10% SDS-PAGE gel along with protein marker, positive
control (histidine-tagged fusion protein), and negative control (bovine
serum albumin, BSA) proteins and transferred to a nitrocellulose membrane
(Bio-Rad Laboratories). The membrane was blocked using 3% (w/v) BSA
in Tris-buffered saline (TBS) for 1 h at room temperature. Subsequently,
the membrane was washed four times with 1× TBS containing 0.05%
Tween-20 (TBST). Next, the membrane was incubated with the anti-His
monoclonal antibody (Santa Cruz Biotechnology) at 1:1000 dilution
for 1 h at room temperature. After washing four times with TBST, the
membrane was incubated for 1 h at room temperature with HRP-conjugated
goat antimouse IgG (Santa Cruz Biotechnology) at 1:5000 dilution.
After washing four times with TBST and once with TBS, the membrane
was incubated with a 3,3′-diaminobenzidine (DAB) substrate
solution (0.5 mg/mL) at room temperature. Color development was stopped
by washing with water, following which the blots were air-dried and
stored.
4
Protein Purity and Size Analysis
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A3C8-GFP-H6 and EM1-GFP-H6
proteins were analyzed to assess their degree of purity by SDS-PAGE
using TGX Stain-Free FastCast gels (BioRad, U.S.). Protein integrity
was also assessed by Western-blot using an anti-His monoclonal antibody
(Santa Cruz Biotechnology, U.S.), and by mass spectrometry (MALDI-TOF).
Additionally, the volume size distribution was determined by dynamic
light scattering (DLS) at 633 nm (Zetasizer Nano ZS, Malvern Instruments
Limited, UK), in 50 μL of protein storage buffer (in of 166 ×
10–3 M NaCO3H and 333 × 10–3 M NaCl). For disassembling, proteins were diluted at 0.5 mg mL–1, and Triton X-100 added to 0.5% was used in order
to visualize their respective building blocks by DLS. Size measurements
were performed in triplicate. Volume data in DLS was representative
of the population size distribution while Intensity data provided
more accurate hydrodynamic sizes.
proteins were analyzed to assess their degree of purity by SDS-PAGE
using TGX Stain-Free FastCast gels (BioRad, U.S.). Protein integrity
was also assessed by Western-blot using an anti-His monoclonal antibody
(Santa Cruz Biotechnology, U.S.), and by mass spectrometry (MALDI-TOF).
Additionally, the volume size distribution was determined by dynamic
light scattering (DLS) at 633 nm (Zetasizer Nano ZS, Malvern Instruments
Limited, UK), in 50 μL of protein storage buffer (in of 166 ×
10–3 M NaCO3H and 333 × 10–3 M NaCl). For disassembling, proteins were diluted at 0.5 mg mL–1, and Triton X-100 added to 0.5% was used in order
to visualize their respective building blocks by DLS. Size measurements
were performed in triplicate. Volume data in DLS was representative
of the population size distribution while Intensity data provided
more accurate hydrodynamic sizes.
5
Recombinant Protein Characterization and NP Analysis
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The integrity of the recombinant proteins was checked by mass spectrometry (MALDI-TOF), TGX (Tris-Glycine eXtended, BioRad, Hercules, CA, USA) Stain-Free acrylamide gels electrophoresis (BioRad, Hercules, CA, USA) and Western Blot analysis using anti-His monoclonal antibody (1:1000; Santa Cruz, ref. 57598, Dallas, TX, USA). Protein concentration wasdetermined by Bradford (Biorad) assay with an albumin (Roche, Basel, Switzerland) standard curve. GFP fluorescence emission (510 nm) was determined on purified proteins with a Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) using an excitation wavelength of 450 nm. The volume and size distribution of NPs were measured by dynamic light scattering (DLS) at 633 nm through a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) using quartz cuvettes.
6
Purification and Characterization of Recombinant Proteins
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Produced proteins were purified by His-tag affinity chromatography using 1 mL HisTrap HP column (GE Healthcare) for T22-HSNBT-H6 and T22-STM-H6 and 1 mL HisTrap excel column (GE Healthcare) for T22-5CTP-H6 through an ÄKTA pure system (GE Healthcare).
Protein separations were made by linear gradient of elution buffer (20 mM Tris, 500 mM NaCl and 500 mM imidazole; pH 8). Purified protein fractions were then dialyzed against sodium carbonate buffer (166 mM NaCO3H; pH 8). Protein purity was analyzed by conventional denaturing SDS-polyacrylamide gel electrophoresis and subsequent Western-blot immunodetection using anti-His monoclonal antibody (Santa Cruz Biotechnology). Finally, protein integrity and glycosylation were determined by MALDI-TOF mass spectrometry.
Protein separations were made by linear gradient of elution buffer (20 mM Tris, 500 mM NaCl and 500 mM imidazole; pH 8). Purified protein fractions were then dialyzed against sodium carbonate buffer (166 mM NaCO3H; pH 8). Protein purity was analyzed by conventional denaturing SDS-polyacrylamide gel electrophoresis and subsequent Western-blot immunodetection using anti-His monoclonal antibody (Santa Cruz Biotechnology). Finally, protein integrity and glycosylation were determined by MALDI-TOF mass spectrometry.
7
Purification and Characterization of His-tagged Proteins
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Proteins were purified as previously described. 20 Briefly, cell pellets were resuspended in Wash buffer (10 mM Tris-HCl, 500 mM NaCl, 10 mM Imidazol pH 8.0) in the presence of protease inhibitors. Cell disruption was performed at 1200 psi using a French Press and the lysate was centrifuged at 15 000g for 45 min. Protein was purified through the His-tag by Immobilized Metal Affinity Chromatography (IMAC) and protein separation was achieved using an imidazole gradient up to 500 mM. Protein peak fractions were collected, dialysed against carbonate buffer (166 mM NaCO 3 H, pH 7.4) and centrifuged to remove insoluble aggregates. Protein integrity was analysed by SDS electrophoresis on TGX Stain-Free gels (Bio-Rad, Hercules, CA, USA) and followed by western blotting using an anti-His monoclonal antibody (Santa Cruz Biotechnology, Inc., Heidelberg, Germany). The protein concentration was determined by using an adapted Bradford's assay. 25 Proteins were found as nanoparticles upon purification, and no specific assembling protocols were applied. Oligomers are presumably formed already in the producing bacteria.
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