The parasite proteins were extracted in reducing sample buffer for SDS-PAGE. Five micrograms of recombinant PvRALP1-Ecto or PvRALP1-Tr protein were loaded into each well and separated by SDS-PAGE under reducing conditions. The separated proteins were transferred to 0.45 μm polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) in a semidry transfer buffer (50 mM Tris, 190 mM glycine, 3.5 mM SDS, 20% methanol) at 400 mA for 40 min using a semidry blotting system (ATTO Corp., Tokyo, Japan). After blocking with 5% skim milk in phosphate-buffered saline containing 0.2% Tween 20 (PBS-T), the membranes were probed with mouse anti-PvRALP1-Ecto and anti-PvRALP1-Tr sera, rabbit anti-PvRALP1-Tr serum, anti-GST monoclonal antibody (Novagen, Madison, WI, USA), anti-penta-His monoclonal antibody (Qiagen), preimmune mouse serum, pooled sera from P. vivax malaria patients or noninfected individuals, all diluted 1:200 in PBS-T. IRDye goat anti-mouse, IRDye goat anti-rabbit, or IRDye goat anti-human sera (LI-COR Biosciences, Lincoln, NE, USA) were used to detect recombinant proteins according to the manufacturer’s instructions. Data were scanned with an Odyssey infrared imaging system (LI-COR Biosciences) and analysed with Odyssey software (LI-COR Biosciences).
Irdye goat anti rabbit
Manufactured by LI COR
Sourced in United States
The IRDye goat anti-rabbit is a secondary antibody used in various immunoassay techniques, such as Western blotting and immunohistochemistry. It is a polyclonal antibody raised in goats and specifically targets rabbit primary antibodies. The antibody is conjugated with an IRDye fluorescent label, which allows for sensitive and quantitative detection of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Irdye goat anti mouse, by LI COR (8 mentions)
Odyssey software, by LI COR (5 mentions)
Odyssey infrared imaging system, by LI COR (5 mentions)
Irdye goat anti human, by LI COR (3 mentions)
Semi dry blotting system, by ATTO Corporation (2 mentions)
Iblot dry blotting system, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
0.45 μm polyvinylidene fluoride membrane, by Merck Group (1 mentions)
0.45 μm pvdf membrane, by Merck Group (1 mentions)
Pvdf membrane, by Cytiva (1 mentions)
Intercept blocking buffer, by LI COR (1 mentions)
Nupage transfer buffer, by Thermo Fisher Scientific (1 mentions)
Nupage 4 to 12 bis tris protein gel, by Thermo Fisher Scientific (1 mentions)
Odyssey fc imaging system, by LI COR (1 mentions)
Protease inhibitor mixture, by Roche (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Malt1, by Cell Signaling Technology (1 mentions)
Ecl donkey anti rabbit igg hrp, by GE Healthcare (1 mentions)
11 protocols using irdye goat anti rabbit
1
Immunoblot Analysis of Recombinant PvRALP1 Proteins
2
Western Blot Analysis of Recombinant Proteins
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Recombinant proteins were analyzed by 13% SDS-PAGE under reducing conditions. The separated proteins were transferred onto a 0.45-μm PVDF membrane (Millipore) in semidry transfer buffer (50 mM Tris, 190 mM glycine, 3.5 mM SDS, 20% methanol) at a constant 400 mA for 40 min using a semidry blotting system (ATTO Corp., Tokyo, Japan). The PVDF membrane (Cytiva, Marlborough, MA) containing recombinant protein was blocked with 5% skim milk in PBS-T (0.5% v/v Tween-20 in 1× PBS) and then incubated with anti-GST antibody (Novagen, Reno, NV) and mouse and rabbit immune sera diluted 1:2,000 in PBS-T. After the primary antibody reaction, the membrane was incubated with the secondary IRDye® goat anti-mouse (1: 5,000 dilution) or IRDye® goat anti-rabbit (1: 5,000) (Li-COR® Bioscience, Lincoln, NE) antibodies to detect antigens. The results were visualized in the Odyssey infrared imaging system and analyzed with Odyssey software (Li-COR® Bioscience) (Lee et al., 2016 (link)).
3
Western Blot Analysis of Protein Lysates
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Cells lines were washed three times with PBS and lysed at 0 °C for 15 min using radio-immunoprecipitation assay (RIPA) buffer (Thermo Scientific, 89900) supplemented with protease inhibitor mixture (Roche, 04693116001). Cell lysate was centrifuged at 15,000 × g for 15 min and supernatant was removed. Protein content in the supernatant was measured using the BCA Protein Assay Kit (Thermo Scientific, 23225). Sodium dodecyl-sulfate polacrylamide gel electrophoresis (SDS-PAGE) analyses were performed in NuPage 4 to 12% Bis-Tris Protein Gels (Invitrogen, NP0335) in Novex 2-(N-morpholino)ethanesulfonic acid (MES) buffer (Invitrogen, B0002). After electrophoresis, proteins were transferred to nitrocellulose membranes in NuPage Transfer buffer (Invitrogen, NP0006) containing 20% methanol (Fisher Scientific, A4524). Membranes were blocked with Intercept Blocking Buffer (LI-COR, 92760001) and incubated with primary antibodies as described in each experimental design and in the figure legends. Then, membranes were washed three times with TBS-T (20 mM Tris HCl pH 7.4, 137 mM NaCl, 2.7 mM KCl, 0.1% Tween-20) and incubated with secondary antibodies IRDye goat anti-rabbit (LI-COR, 92632211) or IRDye goat anti-mouse (LI-COR, 92568070). Western blots were imaged in the Odyssey Fc Imaging System (LI-COR).
4
Immunoblot Analysis of Cell Signaling
5
SDS-PAGE and Western Blot Analysis
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SDS-PAGE
analysis was performed on Novex 10%
Tris-glycine gels (Invitrogen) using 1× Novex Tris-Glycine SDS
running buffer or on NuPAGE 4–12% Bis-Tris gels using 1×
MOPS running buffer at 80 V for 3 to 4 h. Protein samples were diluted
1:1 in Laemmli sample buffer with 5% β-mercaptoethanol (BioRad).
For western blot, gels were incubated at room temperature for 20 min
in 100 mL of equilibration buffer (2× NuPAGE transfer buffer,
10% methanol, and 1:1000 NuPAGE antioxidant). Proteins were transferred
to PVDF using the iBlot dry blotting system (Invitrogen) using program
P3 for 12 min. Immunoblotting was performed according standard protocols
using HRP-conjugated rabbit anti-goat IgG for colorometric visualization
or rabbit anti-CBP (Calmodulin-binding protein, Genscript) followed
by IRDye goat anti-rabbit (LI-COR) for membranes visualized with a
LI-COR Odyssey infrared scanner.
analysis was performed on Novex 10%
Tris-glycine gels (Invitrogen) using 1× Novex Tris-Glycine SDS
running buffer or on NuPAGE 4–12% Bis-Tris gels using 1×
MOPS running buffer at 80 V for 3 to 4 h. Protein samples were diluted
1:1 in Laemmli sample buffer with 5% β-mercaptoethanol (BioRad).
For western blot, gels were incubated at room temperature for 20 min
in 100 mL of equilibration buffer (2× NuPAGE transfer buffer,
10% methanol, and 1:1000 NuPAGE antioxidant). Proteins were transferred
to PVDF using the iBlot dry blotting system (Invitrogen) using program
P3 for 12 min. Immunoblotting was performed according standard protocols
using HRP-conjugated rabbit anti-goat IgG for colorometric visualization
or rabbit anti-CBP (Calmodulin-binding protein, Genscript) followed
by IRDye goat anti-rabbit (LI-COR) for membranes visualized with a
LI-COR Odyssey infrared scanner.
6
Pv50 Protein Characterization by Western Blot
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The recombinant Pv50 and P. vivax schizont-stage parasite lysates were separated by 12% SDS-PAGE and then stained with Coomassie brilliant blue. For western blot analysis, recombinant proteins were electrophoretically transferred to polyvinylfluoride membranes (Millipore Corp., Billerica, MA, USA), and incubated with blocking buffer (5% non-fat milk in PBS containing 0.2% Tween 20, PBS/T) for 1 h at 37 °C. After blocking, penta anti-His antibody, mouse immune sera, rabbit immune sera, or mixed patient serum were diluted by 200-fold with PBS/T, and the specific quality of the His-tagged recombinant protein and immune serum was examined using the secondary antibody IRDye® goat anti-mouse (1:10,000 dilution), IRDye® goat anti-rabbit (1:20,000 dilution), or IRDye® goat anti-human (1:20,000) (LI-COR Biosciences, Lincoln, NE, USA). The fluorescence signals from the reaction were scanned on an Odyssey infrared imaging system (LI-COR Biosciences) and analyzed with Odyssey software (Li-Cor Biosciences).
7
Protein Extraction and Western Blot Analysis
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Total protein was extracted from N2A cells using RIPA with 1:1,000 protease inhibitor cocktail (Sigma-Aldrich) and 1:500 phenylmethyl sulphonyl fluoride. Cells were lysed directly on the culture plate with 100–150 μl RIPA and scraped off with a cell scraper. The lysate was transferred to a micro-centrifuge tube and sonicated for five cycles, each comprising 30 s of sonication with 30 s intervals. The resulting mixture was centrifuged for 15 min at 14,000g to separate the protein mixture (supernatant) from cellular debris (pellet). Proteins (20–40 μg) were resolved on SDS–PAGE and transferred onto a nitrocellulose membrane (Bio-rad). Membranes were blocked in 3% BSA for 1 h, and then incubated in primary (overnight at 4 °C)and secondary (1 h at room temperature) antibodies. They were scanned using Odessey IR scanner (Li-Cor Bioscience), and band intensity was determined and quantified using image analysis software (ImageJ). The following antibodies were used: primary-HDAC9 (Abcam, ab59718), PP1γ (Millipore, 07-1218), GAPDH (Abcam, ab9485), beta actin (Abcam, ab8226); secondary-anti-mouse IRDye goat anti-mouse (LI-COR, 925-32210) and IRDye goat anti-rabbit (LI-COR, 925-32211). Original blots are shown in (Supplementary Figs 15 and 16 ).
8
Plasma Membrane Protein Solubilization and Immunoblotting
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Three micrograms of plasma membrane
protein was combined with 1% digitonin in membrane-solubilization
buffer (50 mM Tris-HCl, 150 mM NaCl, and 1 mM EDTA, pH 7.5) as previously
described.43 (link) NativePAGE sample buffer (Invitrogen)
was added to the supernatant to a final concentration of 1×,
and NativePAGE 5% G-250 sample additive was added to a final concentration
of 0.06%. Samples were run on NativePAGE Novex 3–12% Bis-Tris
gels at 150 V for 20 min with dark blue cathode buffer followed by
45 min with light blue cathode buffer. Gels were washed for 20 min
in equilibration buffer (80 mL of ddH20, 10 mL of 20×
transfer buffer, 10 mL of methanol, and 100 μL of NuPage antioxidant),
transferred to Immobilon-FL membranes (Millipore) using an iBlot dry
blotting system (Invitrogen). Membrane was allowed to dry 30 min.
Immunoblotting was performed according to a standard protocol using
rabbit anti-CBP and IRDye goat anti-rabbit (LI-COR). Membranes were
visualized with a LI-COR Odyssey infrared-imaging system.
protein was combined with 1% digitonin in membrane-solubilization
buffer (50 mM Tris-HCl, 150 mM NaCl, and 1 mM EDTA, pH 7.5) as previously
described.43 (link) NativePAGE sample buffer (Invitrogen)
was added to the supernatant to a final concentration of 1×,
and NativePAGE 5% G-250 sample additive was added to a final concentration
of 0.06%. Samples were run on NativePAGE Novex 3–12% Bis-Tris
gels at 150 V for 20 min with dark blue cathode buffer followed by
45 min with light blue cathode buffer. Gels were washed for 20 min
in equilibration buffer (80 mL of ddH20, 10 mL of 20×
transfer buffer, 10 mL of methanol, and 100 μL of NuPage antioxidant),
transferred to Immobilon-FL membranes (Millipore) using an iBlot dry
blotting system (Invitrogen). Membrane was allowed to dry 30 min.
Immunoblotting was performed according to a standard protocol using
rabbit anti-CBP and IRDye goat anti-rabbit (LI-COR). Membranes were
visualized with a LI-COR Odyssey infrared-imaging system.
9
Recombinant Protein Characterization via SDS-PAGE and Western Blot
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rPv32 was separated using 12% SDS-PAGE after denaturation with the reducing agent β-mercaptoethanol in sample buffer and then stained with Coomassie brilliant blue. For western blot analysis, recombinant proteins were transferred electrophoretically to PVDF membranes (Millipore Corp., Bedford, MA, USA) and incubated with blocking buffer (5% non-fat milk in PBS containing 0.2% Tween 20 and PBS/T) for 1 h at 37 °C. After blocking, either anti-GST antibody, mouse and rabbit immune sera or mixed patient sera was diluted with PBS/T 200 times. Secondary IRDye® goat anti-mouse (1:10,000 dilution), IRDye® goat anti-rabbit (1:20,000 dilution) or IRDye® goat anti-human (1:20,000) (LI-COR® Bioscience, Lincoln, NE, USA) were used to detect GST-tagged recombinant protein and immune serum of a specific quality. Sera from healthy people from the ROK and PBS-immunized rabbit serum were used as controls. Data were scanned with an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA) and analyzed by Odyssey software (LI-COR, Inc.).
10
Western Blot Analysis of Phospho-Slt2 and Control Proteins
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For Western blot analysis, cell equivalents of an OD600 of 3 were harvested at 24 h after inoculation, and cell extracts were obtained from chemical lysis as described in.91 (link) Proteins were collected by centrifugation and resuspended in 75 μL 1× loading buffer (125 mM Tris-HCl, adjusted to pH 6.8; 20% glycerol; 3% SDS; 2% DTT; 0.1% bromophenol blue), and heated to 95°C for 10 min. Samples were centrifuged at 13,000 rpm for 12 s and 10 μL or 15 μL of the supernatant was used for standard SDS-PAGE. Immunoblotting followed standard procedures, with transfer of proteins to a 0.45 μm nitrocellulose membrane and probing with antibodies against phospho-Slt2 (Phospho-p44/42 MAPK, cell signaling, #9101, 1:1000), actin (α-Yeast act1 Goat monoclonal antibody, a kind gift from Prof. John Cooper, Washington University, 1:2000), (glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Life Technologies, MA515738, 1:5000), or VDAC/porin (Abcam, ab110326, 1:5000). As secondary antibodies, we used IRDye goat anti-mouse (Licor, 926–68070, 1:20,000) or IRDye goat anti-rabbit (Licor, 928-40028, 1:20,000) as listed in the key resources table . Signals were recorded with Odyssey Glx, with automatically determined exposure times. Quantitative analysis of western blots was performed using image studio software.
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