Membrane proteins were biotinylated and purified using the Pierce Cell Surface Protein Isolation Kit according to the manufacturer’s instructions (Pierce, Thermo Scientific, Rockford, IL, USA). Western blot analysis was performed using a mouse monoclonal primary antibody against V5 (Invitrogen) and then a polyclonal goat anti-mouse immunoglobulin/HRP (Dako) as secondary antibody. The membranes were incubated with the Immobilon Forte Western HRP substrate according to the manufacturer’s instructions (Merck Millipore, Burlington, MA, USA) and digitized for pattern analysis using the GeneGnome system (Syngene), Paris, France).
Polyclonal goat anti mouse immunoglobulin hrp
Manufactured by Agilent Technologies
Sourced in Denmark
Polyclonal goat anti-mouse immunoglobulin/HRP is a secondary antibody conjugated with horseradish peroxidase (HRP). It is used to detect and visualize mouse primary antibodies in various immunoassays and immunochemical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Merck Group (4 mentions)
Trans blot, by Bio-Rad (4 mentions)
Nitrocellulose membrane, by Agilent Technologies (2 mentions)
Mab against flag tag, by GenScript (2 mentions)
Genegnome system, by Syngene (1 mentions)
Immobilon forte western hrp substrate, by Merck Group (1 mentions)
Pierce ecl plus western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Sequi blot pvdf membrane, by Bio-Rad (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
6 protocols using polyclonal goat anti mouse immunoglobulin hrp
1
Membrane Protein Biotinylation and Western Blot
2
Investigating anxA5-Stx2 Binding Interaction
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A direct interaction between anxA5 and Stx2 was assessed. An activated Sequi-Blot PVDF membrane (Bio-Rad Laboratories, Hercules, CA, USA) was coated with anxA5 (1 or 10 μg) or Stx2 (positive control, 1 μg, Phoenix Lab) or bovine serum albumin (negative control, 1% BSA, Sigma-Aldrich) and allowed to dry. The PVDF membrane was blocked with 1% BSA for 1 h at RT. The membrane was washed three times with 0.05% PBS-Tween (PBS-T, Medicago, Uppsala, Sweden) and incubated with Stx2 diluted in 1% BSA (200 ng/mL) for 1 h at RT. After washing, the membrane was incubated with mouse anti-Stx2 (1:100, Santa Cruz Biotechnology, Dallas, TX) for 1 h at RT, washed and further incubated with polyclonal goat anti-mouse immunoglobulin HRP (1:1000, Dako, Glostrup, Denmark) for 1 h at RT. The membrane was washed and developed using Pierce ECL Plus Western Blotting Substrate (Thermo Fisher Scientific) according to manufacturer’s protocol and visualized using a GelDoc Touch Imaging System (Bio-Rad Laboratories).
3
Western Blot Analysis of TgGRA8 Immunoreactivity
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The immunoreactivity of the expressed TgGRA8 protein was determined by Western blotting using SDS-PAGE and then electrotransferred (Trans-blot, Bio-Rad) onto a nitrocellulose membrane (Millipore, USA).
The membrane was washed three times with PBS, blocked with 5% skim milk, and then incubated at 37 ℃ for 1 h. After incubation, the membrane was washed three times with PBS containing 0.01% Tween 20
(PBS-T) and rinsed with PBS. The TgGRA8 protein in nitrocellulose membrane was probed using known reference positive and negative goat sera (diluted 1:250 in 5% skim milk) kept in our laboratory and incubated at 37℃ for 1 h. The membrane was washed three times with PBS-T and incubated with polyclonal goat anti-mouse immunoglobulin/HRP (Dako, Denmark) diluted 1:2000 in blocking buffer. The protein band was developed according to peroxidase activity using 3,3′,5,5′-tetramethylbenzidine (KPL, Gaithersburg, MD, USA).
The membrane was washed three times with PBS, blocked with 5% skim milk, and then incubated at 37 ℃ for 1 h. After incubation, the membrane was washed three times with PBS containing 0.01% Tween 20
(PBS-T) and rinsed with PBS. The TgGRA8 protein in nitrocellulose membrane was probed using known reference positive and negative goat sera (diluted 1:250 in 5% skim milk) kept in our laboratory and incubated at 37℃ for 1 h. The membrane was washed three times with PBS-T and incubated with polyclonal goat anti-mouse immunoglobulin/HRP (Dako, Denmark) diluted 1:2000 in blocking buffer. The protein band was developed according to peroxidase activity using 3,3′,5,5′-tetramethylbenzidine (KPL, Gaithersburg, MD, USA).
4
Recombinant TgGRA8 Protein Immunoblotting
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Five micrograms of recombinant TgGRA8 was resolved by 12% SDS-PAGE and then electrotransferred (Trans-blot, Bio-Rad) onto a nitrocellulose membrane (Millipore, USA). The membrane was washed three times with PBS, blocked with 5% skim milk, and then incubated at 37 °C for 1 h with constant shaking. After incubation, the membrane was washed three times with PBS containing 0.01% Tween 20 (PBS-T) and rinsed with PBS. The TgGRA8 protein in nitrocellulose membrane was probed using antibody or known reference positive and negative goat sera (diluted 1:250 in 5% skim milk) kept in our laboratory and incubated at 37 °C for 1 h with constant shaking. The monoclonal antibody (mAb) against Flag-tag (GenScript, USA) was diluted 1:1000, while polyclonal mouse anti-goat immunoglobulin/HRP (Dako, Denmark) was diluted 1:2000 in blocking buffer. After incubation, the membrane was washed three times with PBS-T. The protein band was developed according to peroxidase activity using 3,3′,5,5′-tetramethylbenzidine (KPL, Gaithersburg, MD, USA).
5
Western Blot Analysis of TgGRA8
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Five micrograms of recombinant TgGRA8 was resolved by 12% SDS-PAGE and then electrotransferred (Trans-blot, Bio-Rad) onto a nitrocellulose membrane (Millipore, USA). The membrane was washed three times with PBS, blocked with 5% skim milk, and then incubated at 37 ℃ for 1 h with constant shaking.
After incubation, the membrane was washed three times with PBS containing 0.01% Tween 20 (PBS-T) and rinsed with PBS. The TgGRA8 protein in nitrocellulose membrane was probed using antibody or known reference positive and negative goat sera (diluted 1:250 in 5% skim milk) kept in our laboratory and incubated at 37℃ for 1 h with constant shaking. The monoclonal antibody (mAb) against Flag-tag (GenScript, USA) was diluted 1:1000, while polyclonal mouse anti-goat immunoglobulin/HRP (Dako, Denmark) was diluted 1:2000 in blocking buffer. After incubation, the membrane was washed three times with PBS-T. The protein band was developed according to peroxidase activity using 3,3′,5,5′tetramethylbenzidine (KPL, Gaithersburg, MD, USA).
After incubation, the membrane was washed three times with PBS containing 0.01% Tween 20 (PBS-T) and rinsed with PBS. The TgGRA8 protein in nitrocellulose membrane was probed using antibody or known reference positive and negative goat sera (diluted 1:250 in 5% skim milk) kept in our laboratory and incubated at 37℃ for 1 h with constant shaking. The monoclonal antibody (mAb) against Flag-tag (GenScript, USA) was diluted 1:1000, while polyclonal mouse anti-goat immunoglobulin/HRP (Dako, Denmark) was diluted 1:2000 in blocking buffer. After incubation, the membrane was washed three times with PBS-T. The protein band was developed according to peroxidase activity using 3,3′,5,5′tetramethylbenzidine (KPL, Gaithersburg, MD, USA).
6
Immunoreactivity Determination of TgGRA8 Protein
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The immunoreactivity of the expressed TgGRA8 protein was determined by Western blotting using SDS-PAGE and then electrotransferred (Trans-blot, Bio-Rad) onto a nitrocellulose membrane (Millipore, USA). The membrane was washed three times with PBS, blocked with 5% skim milk, and then incubated at 37 ℃ for 1 h. After incubation, the membrane was washed three times with PBS containing 0.01% Tween 20
(PBS-T) and rinsed with PBS. The TgGRA8 protein in nitrocellulose membrane was probed using known reference positive and negative goat sera (diluted 1:250 in 5% skim milk) kept in our laboratory and incubated at 37℃ for 1 h. The membrane was washed three times with PBS-T and incubated with polyclonal mouse anti-goat immunoglobulin/HRP (Dako, Denmark) diluted 1:2000 in blocking buffer. The protein band was developed according to peroxidase activity using 3,3′,5,5′-tetramethylbenzidine (KPL, Gaithersburg, MD, USA).
(PBS-T) and rinsed with PBS. The TgGRA8 protein in nitrocellulose membrane was probed using known reference positive and negative goat sera (diluted 1:250 in 5% skim milk) kept in our laboratory and incubated at 37℃ for 1 h. The membrane was washed three times with PBS-T and incubated with polyclonal mouse anti-goat immunoglobulin/HRP (Dako, Denmark) diluted 1:2000 in blocking buffer. The protein band was developed according to peroxidase activity using 3,3′,5,5′-tetramethylbenzidine (KPL, Gaithersburg, MD, USA).
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