Activated gametocyte and ookinete samples were isolated as described below. WT or SAS6L-GFP samples were then purified using a GFP-Trap kit to immunoprecipitate GFP-fusion protein (Chromotek). After the addition of Laemmli sample buffer, the samples were boiled and loaded on a 4–12% SDS-polyacrylamide gel. Samples were subsequently transferred to nitrocellulose membranes (Amersham Biosciences) with immunoblotting performed using the Western Breeze Chemiluminescent Anti-Rabbit kit (Invitrogen) and anti-GFP polyclonal antibody (Invitrogen) at a concentration of 1:1250, according to the manufacturer’s instructions.
Anti gfp polyclonal antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Anti-GFP polyclonal antibody is a laboratory reagent that recognizes and binds to green fluorescent protein (GFP). It can be used to detect the presence of GFP-tagged proteins in various applications, such as Western blotting, immunoprecipitation, and immunocytochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Cytiva (4 mentions)
Western breeze chemiluminescent anti rabbit kit, by Thermo Fisher Scientific (3 mentions)
Gfp trap kit, by Proteintech (2 mentions)
Gfp fusion protein, by Proteintech (2 mentions)
Western breeze chemiluminescence anti rabbit kit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor labeled secondary antibody, by Thermo Fisher Scientific (1 mentions)
Irdye 680rd goat anti mouse igg h l, by LI COR (1 mentions)
Irdye 800cw donkey anti rabbit igg h l, by LI COR (1 mentions)
Anti flag polyclonal ab, by Merck Group (1 mentions)
0.45 μm nitrocellulose membrane, by Merck Group (1 mentions)
Novex tris glycine gel, by Thermo Fisher Scientific (1 mentions)
Pierce fast blocking buffer, by Thermo Fisher Scientific (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Diva software, by BD (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
8 protocols using anti gfp polyclonal antibody
1
GFP-Fusion Protein Immunoprecipitation
2
Purification and Analysis of Parasite Proteins
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Parasite-infected red blood cells were placed in schizont culture medium (RPMI 1640, FCS 1:10, Penicillin/Streptomycin 1:100) for 24 h at 37°C and parasites were allowed to mature to schizonts, which were purified the following day on a 60% v/v NycoDenz (in PBS) gradient, harvested from the interface and washed (stock solution: 27.6% w/v NycoDenz in 5 mM Tris-HCl, pH 7.20, 3 mM KCl, 0.3 mM EDTA). After the addition of Laemmli sample buffer to the cells, the samples were boiled and electrophoresed on a 4–12% SDS-polyacrylamide gel. Resolved proteins were subsequently transferred to nitrocellulose membranes (Amersham Biosciences) and immunoblotting performed using the Western Breeze Chemiluminescent Anti-Rabbit kit (Invitrogen) and anti-GFP polyclonal antibody (Invitrogen) at a concentration of 1:1250, according to the manufacturer's instructions.
A schizont lysate was also used for immunoprecipitation with a GFP-Trap®_A Kit (Chromotek) following the manufacturer's instructions. The GFP-Trap®_A beads were equilibrated with dilution buffer and proteins bound from the parasite lysate by slow mixing at 4°C for 2 h. The beads were then harvested by centrifugation at 1200g for 2 min and washed twice with dilution buffer. The beads with bound proteins were then treated with trypsin and released peptides were analyzed by tandem mass spectrometry.
A schizont lysate was also used for immunoprecipitation with a GFP-Trap®_A Kit (Chromotek) following the manufacturer's instructions. The GFP-Trap®_A beads were equilibrated with dilution buffer and proteins bound from the parasite lysate by slow mixing at 4°C for 2 h. The beads were then harvested by centrifugation at 1200
3
Isolation and Immunoblotting of GFP-Fusion Proteins
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Schizont, gametocyte and ookinete samples were isolated as described below. WT-GFP or CYC3-GFP samples were then purified using a GFP-Trap kit to immunoprecipitate GFP-fusion protein (Chromotek). After the addition of Laemmli sample buffer, the samples were boiled and an equal concentration of total protein was loaded on a 4–12% SDS-polyacrylamide gel. Samples were subsequently transferred to nitrocellulose membranes (Amersham Biosciences) and immunoblotting performed using the Western Breeze Chemiluminescent Anti-Rabbit kit (Invitrogen) and anti-GFP polyclonal antibody (Invitrogen) at a concentration of 1:1250, according to the manufacturer's instructions.
4
Western Blotting Protocol for Purified Schizonts
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For western blotting, purified schizonts were lysed using lysis buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 1% NP-40 and 1% sarkosyl). The samples were boiled for 10 min after adding Laemmli sample buffer to the lysed cells. The sample was centrifuged at 13,500 g for 5 min and electrophoresed on a 4–12% SDS–polyacrylamide gel. Subsequently, resolved proteins were transferred to nitrocellulose membrane (Amersham Biosciences) and immunoblotting was performed using the Western Breeze Chemiluminescence anti-rabbit kit (Invitrogen) and anti-GFP polyclonal antibody (Invitrogen) at a dilution of 1:1250, according to the manufacturer's instructions.
5
Antibody Characterization for Protein Signaling
6
SDS-PAGE and Western Blot Analysis of GvpC Protein
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The methods used were those previously described [7 (link), 17 (link)]. Briefly, the SDS-PAGE analysis was performed using the precast Novex® Tris-glycine gels (4–20%, Invitrogen, Carlsbad, CA, USA). Proteins were then transferred to 0.45 μm nitrocellulose membranes (Millipore Corp., Boston, MA). Membranes were blocked for 30 min in Pierce Fast Blocking Buffer (Thermo Fisher Scientific), incubated in blocking buffer supplemented with Anti-GFP Polyclonal Antibody (Thermo Fisher Scientific) or Anti-GvpC antibodies (GenScript, USA), followed by three washing steps with TBST and then incubation with Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Invitrogen, Catalog # A-11008).
7
Quantifying Malaria Parasitemia by Flow Cytometry
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To introduce a large-scale parasitemia monitoring method, we employed nuclear staining followed by a flow cytometry analysis. Infected red blood cells were pelleted, washed with 1× phosphate buffered saline (PBS), and fixed in a solution of 4% paraformaldehyde and 0.025% glutaraldehyde in PBS for 30 min at room temperature (RT). The fixed cells were subsequently washed twice with PBS (600×g, 3 min) and stored at 4°C for up to three weeks. To analyze parasitemia, the cells were stained with 0.02mM Ethidium Homodimer 1 (EthD-1, Biotinum) diluted in PBS for 30 min at RT. Afterwards, the stained samples were washed twice (600×g, 3 min) with PBS and analyzed using a FACS CantoII flow cytometer and Diva software provided by BD Biosciences, including the determination of Median Fluorescence Intensity (MFI) values for the GFP signal. When needed, flow cytometry was utilized to determine the GFP signal from transgenic parasites. Fixed parasites samples stained with EthD-1 were incubated with an Anti-GFP Polyclonal Antibody conjugated with Alexa Fluor™ 488 (Thermofisher), diluted 100× in PBS for 30 min. The samples were then washed twice with PBS (600×g, 3 min) and analyzed in the same manner as stated above.
8
Conjugation of anti-GFP Antibody with CuNP
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Anti GFP polyclonal antibody from Thermo Fischer Scientific and GFP Recombinant Aequorea Victoria Protein from Invitrogen have been used for conjugation study. 10 μg ml−1 anti GFP antibody and 0.0134 mg ml−1 CuNP was used for coating. The antibody was mixed well with the CuNP, kept at 4 °C for a day, washed 3 times by centrifugation to get rid of the unbound antibody.
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