Oligonucleotides were synthesized by Integrated DNA Technologies (Coralville, CA). The QuikChange II mutagenesis kit was obtained from Agilent Technologies (Santa Clara, CA). [D-glucose-14C-(U)]Lactose was purchased from Moravek Biochemicals (Brea, CA). Tetramethylrhodamine 5-maleimide (TMRM) was purchased from Invitrogen (Carlsbad, CA). p-Nitrophenyl α-D-galactopyranoside (α-NPG) was obtained from Sigma-Aldrich (St. Louis, MO). α-D-Melibiose was obtained from CHEM-IMPEX (Wood Dale, IL), and immobilized monomeric avidin was purchased from Thermo Scientific (Rockford, IL). The penta-His antibody–horseradish peroxidase (HRP) conjugate was obtained from Qiagen (Hilden, Germany). All other materials were reagent grade and obtained from commercial sources.
Penta his antibody horseradish peroxidase hrp conjugate
Manufactured by Qiagen
Sourced in Germany
The Penta-His antibody–horseradish peroxidase (HRP) conjugate is a laboratory reagent used for the detection of proteins tagged with a His-tag. It consists of an anti-His-tag antibody conjugated to the enzyme horseradish peroxidase, which can be used in various immunoassay techniques.
Automatically generated - may contain errors
Lab products found in correlation
Quikchange 2 mutagenesis kit, by Agilent Technologies (2 mentions)
D glucose 14c u lactose, by Moravek Biochemicals (2 mentions)
Blue dextran, by Merck Group (2 mentions)
Aldolase, by Merck Group (2 mentions)
Carbonic anhydrase, by Merck Group (2 mentions)
Conalbumin, by Merck Group (2 mentions)
Kta purifier, by Cytiva (2 mentions)
Amersham protran 0.45 μm nc, by GE Healthcare (2 mentions)
Hiload 16 600 superdex 75 pg, by GE Healthcare (2 mentions)
Rnase a, by Merck Group (2 mentions)
Immobilized monomeric avidin, by Thermo Fisher Scientific (1 mentions)
Oligonucleotide, by Integrated DNA Technologies (1 mentions)
Melibiose, by Thermo Fisher Scientific (1 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Lactose, by Merck Group (1 mentions)
Restriction enzyme, by New England Biolabs (1 mentions)
4 protocols using penta his antibody horseradish peroxidase hrp conjugate
1
Synthesis and Characterization of Labeled Biomolecules
2
Site-Directed Mutagenesis Utilizing Oligonucleotides
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Oligonucleotides for site-directed mutagenesis
were synthesized by Integrated DNA Technologies, Inc. (Coralville,
IA). A QuikChange II mutagenesis kit was obtained from Agilent Technologies,
Inc. (Santa Clara, CA). Restriction enzymes were purchased from New
England Biolabs Inc. (Ipswich, MA). Lactose and p-nitrophenyl α-d -galactopyranoside (NPG) were from
Sigma-Aldrich (St. Louis, MO). Melibiose was obtained from Acros Organics.
[d -glucose-14C(U)]Lactose was from Moravek Biochemicals,
Inc. (Brea, CA). The penta-His antibody–horseradish peroxidase
(HRP) conjugate was from Qiagen (Hilden, Germany). Supersignal West
Pico Chemiluminescent substrate kits for Western blotting were from
Pierce (Rockford, IL). Micro BCA protein assay kits were from Thermo
Scientific (Rockford, IL).
were synthesized by Integrated DNA Technologies, Inc. (Coralville,
IA). A QuikChange II mutagenesis kit was obtained from Agilent Technologies,
Inc. (Santa Clara, CA). Restriction enzymes were purchased from New
England Biolabs Inc. (Ipswich, MA). Lactose and p-nitrophenyl α-
Sigma-Aldrich (St. Louis, MO). Melibiose was obtained from Acros Organics.
[
Inc. (Brea, CA). The penta-His antibody–horseradish peroxidase
(HRP) conjugate was from Qiagen (Hilden, Germany). Supersignal West
Pico Chemiluminescent substrate kits for Western blotting were from
Pierce (Rockford, IL). Micro BCA protein assay kits were from Thermo
Scientific (Rockford, IL).
3
Analysis of Purified Proteins
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The molecular masses of the purified proteins in a denatured state were analyzed by SDS-PAGE (53 (link), 54 ). Immunodetection of the His10-tagged proteins was performed via Western blotting (55 (link)) using a nitrocellulose blotting membrane (Amersham Protran 0.45-μm NC, GE Healthcare, Germany) and a Penta-His horseradish peroxidase (HRP) conjugate antibody (Qiagen, Germany).
Native sizes were analyzed via size exclusion chromatography on a HiLoad 16/600 Superdex 75 pg (GE Healthcare, Germany) using 10 mM Tris-HCl, pH 8, with 300 mM NaCl at a flow rate of 1 ml min−1 with an ÄKTA purifier (Amersham Pharmacia Biotech, UK). Calibration was performed with standard proteins of known sizes (RNase A, 13,700 Da; carbonic anhydrase, 29,000 Da; conalbumin, 75,000 Da; aldolase, 158,000 Da; blue dextran; Sigma-Aldrich, Steinheim, Germany) under similar conditions. The resulting standard curve was used for size determination of respective elution peaks of StyI and StyJ. Peak fractions were collected and analyzed by SDS-PAGE and Western blotting.
Final concentrations of pure proteins were calculated from the respective absorptions at 280 nm, applying molar extinction coefficients of 42,860 M−1 cm−1 (StyI) and 48,150 M−1 cm−1 (StyJ) as well as molecular weights of 29,836.3 g mol−1 (StyI) and 30,095.6 g mol−1 (StyJ) as predicted by Expasy ProtParam (56 (link)).
Native sizes were analyzed via size exclusion chromatography on a HiLoad 16/600 Superdex 75 pg (GE Healthcare, Germany) using 10 mM Tris-HCl, pH 8, with 300 mM NaCl at a flow rate of 1 ml min−1 with an ÄKTA purifier (Amersham Pharmacia Biotech, UK). Calibration was performed with standard proteins of known sizes (RNase A, 13,700 Da; carbonic anhydrase, 29,000 Da; conalbumin, 75,000 Da; aldolase, 158,000 Da; blue dextran; Sigma-Aldrich, Steinheim, Germany) under similar conditions. The resulting standard curve was used for size determination of respective elution peaks of StyI and StyJ. Peak fractions were collected and analyzed by SDS-PAGE and Western blotting.
Final concentrations of pure proteins were calculated from the respective absorptions at 280 nm, applying molar extinction coefficients of 42,860 M−1 cm−1 (StyI) and 48,150 M−1 cm−1 (StyJ) as well as molecular weights of 29,836.3 g mol−1 (StyI) and 30,095.6 g mol−1 (StyJ) as predicted by Expasy ProtParam (56 (link)).
4
Analysis of Purified Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The molecular masses of the purified proteins in a denatured state were analyzed by SDS-PAGE (53 (link), 54 ). Immunodetection of the His10-tagged proteins was performed via Western blotting (55 (link)) using a nitrocellulose blotting membrane (Amersham Protran 0.45-μm NC, GE Healthcare, Germany) and a Penta-His horseradish peroxidase (HRP) conjugate antibody (Qiagen, Germany).
Native sizes were analyzed via size exclusion chromatography on a HiLoad 16/600 Superdex 75 pg (GE Healthcare, Germany) using 10 mM Tris-HCl, pH 8, with 300 mM NaCl at a flow rate of 1 ml min−1 with an ÄKTA purifier (Amersham Pharmacia Biotech, UK). Calibration was performed with standard proteins of known sizes (RNase A, 13,700 Da; carbonic anhydrase, 29,000 Da; conalbumin, 75,000 Da; aldolase, 158,000 Da; blue dextran; Sigma-Aldrich, Steinheim, Germany) under similar conditions. The resulting standard curve was used for size determination of respective elution peaks of StyI and StyJ. Peak fractions were collected and analyzed by SDS-PAGE and Western blotting.
Final concentrations of pure proteins were calculated from the respective absorptions at 280 nm, applying molar extinction coefficients of 42,860 M−1 cm−1 (StyI) and 48,150 M−1 cm−1 (StyJ) as well as molecular weights of 29,836.3 g mol−1 (StyI) and 30,095.6 g mol−1 (StyJ) as predicted by Expasy ProtParam (56 (link)).
Native sizes were analyzed via size exclusion chromatography on a HiLoad 16/600 Superdex 75 pg (GE Healthcare, Germany) using 10 mM Tris-HCl, pH 8, with 300 mM NaCl at a flow rate of 1 ml min−1 with an ÄKTA purifier (Amersham Pharmacia Biotech, UK). Calibration was performed with standard proteins of known sizes (RNase A, 13,700 Da; carbonic anhydrase, 29,000 Da; conalbumin, 75,000 Da; aldolase, 158,000 Da; blue dextran; Sigma-Aldrich, Steinheim, Germany) under similar conditions. The resulting standard curve was used for size determination of respective elution peaks of StyI and StyJ. Peak fractions were collected and analyzed by SDS-PAGE and Western blotting.
Final concentrations of pure proteins were calculated from the respective absorptions at 280 nm, applying molar extinction coefficients of 42,860 M−1 cm−1 (StyI) and 48,150 M−1 cm−1 (StyJ) as well as molecular weights of 29,836.3 g mol−1 (StyI) and 30,095.6 g mol−1 (StyJ) as predicted by Expasy ProtParam (56 (link)).
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