Cells were first stained with antibodies directed against extracellular markers (CD45, CD11b, Ly6G) and then permeabilized with Cytofix/Cytoperm and Permwash buffer (BD Biosciences). Intracellular staining of F-actin was performed with Phalloidin, Fluorescein Isothiocyanate Labeled (Sigma-Aldrich, ref P5282), whereas α-tubulin was labeled with α-Tubulin antibody (clone 11H10, Cell Signaling, ref 2125) followed by secondary an anti-rabbit IgG antibody coupled to Alexa Fluor 555 (ThermoFisher, ref A27039). Phospho-p42/44 MAPK ERK1/2 (Thr 202/Tyr 204) (Cell signaling, ref 4370) followed by secondary an anti-rabbit IgG antibody coupled to Alexa Fluor 555 (ThermoFisher, ref A27039) was used to detect Phospho-p42/44 MAPK ERK1/2. p-p38 MAPK (Thr 180/Tyr 182) coupled to PE (cell Signaling 6908), was used to detect phospho-p38 MAPK.
Anti rabbit igg antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Anti-rabbit IgG antibody is a laboratory reagent used to detect and quantify the presence of rabbit immunoglobulin G (IgG) in samples. It is a polyclonal antibody that binds specifically to rabbit IgG proteins. This antibody can be used in various immunoassay techniques, such as ELISA, Western blotting, and immunohistochemistry, to measure and analyze rabbit IgG levels in research and diagnostic applications.
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Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Horseradish peroxidase conjugated anti mouse igg antibody, by Thermo Fisher Scientific (3 mentions)
Anti mouse igg antibody, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Ecl system, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Perm wash buffer, by BD (1 mentions)
Phospho p42 44 mapk erk1 2, by Cell Signaling Technology (1 mentions)
Nytran, by Cytiva (1 mentions)
Whatman, by Cytiva (1 mentions)
Ecl substrate, by Thermo Fisher Scientific (1 mentions)
Nylon membrane, by GE Healthcare (1 mentions)
Hrp conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Rabbit anti m6a antibody, by Synaptic Systems (1 mentions)
Acrylamide bis acrylamide solution, by Bio-Rad (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Clarity max western ecl substrate, by Bio-Rad (1 mentions)
Temed, by Bio-Rad (1 mentions)
Ammonium persulfate aps, by Bio-Rad (1 mentions)
29 protocols using anti rabbit igg antibody
1
Multicolor Flow Cytometry Analysis
2
Immunoblotting for N6-Methyladenine Detection
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Immunoblotting was performed and normalized as previously described (Wu et al., 2016 (link); Xie et al., 2018 (link); Kweon et al., 2019 (link)). Briefly, DNA was denatured at 95°C for 10 min, flash-cooled on ice and neutralized with 10% (v/v) of 6.6M Ammonium Acetate. Samples (40 ng, 4 μl per sample) were spotted on a nylon membrane (Whatman Nytran SuperCharge; Sigma), air-dried for 10 min and UV-crosslinked for 90 s (InGenius syngene bio imaging). Membranes were then blocked (5% non-fat milk, 1% BSA in 0.1% PBST) for 1 h at room temperature (RT) followed by incubation with the primary anti-N6-methyladenine antibody (Synaptic Systems) diluted 1:1000 in blocking solution overnight, at 4°C. After washing three times with 0.1% PBST, membranes were incubated with anti-rabbit IgG antibody (Thermo Fisher Scientific) diluted 1:5000 in blocking solution for 2 h at RT.
3
SDS-PAGE and Western Blot Analysis
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The original propagated virus preparations were denatured and fractionated on a discontinuous SDS-PAGE gel alongside a series of molecular weight markers of known size and visualized with Coomassie Blue staining [1 , 3 , 29 (link)]. The molecular mass of the viral proteins was estimated from a calibration curve established by the migration distances of the molecular weight markers [8 ]. The proteins were transferred to nitrocellulose membrane (Western blot) according to Allen and Ball [1 ] (see also [29 (link)]). The Western blots were incubated for 16 h at room temperature with a 1/1000 dilution of primary antibody in phosphate-buffered saline phosphate (PBS) containing 1% Tween-20 and 5% fat-free powdered milk (PBS-TM). The membrane was washed 6 × 7 min with PBS-TM and incubated 2 h at room temperature with a 1/4000 dilution of an anti-rabbit-IgG antibody covalently linked to alkaline phosphatase (ThermoFisher: Waltham, MA, USA) in PBSTM. The membranes were washed 5 × 7 min with PBS and developed with 5% nitroblue tetrazolium and 5% 5-Bromo-4-Chloro-3-Indolyl Phosphate in 0.1M TRIS(9.5)/0.1M NaCl/50 mM MgCl2 [1 ].
4
Antibody-based Detection of RNA Modifications
5
Western Blot Analysis of TET1 and TET3
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Protein lysates were prepared using AllPrep DNA/RNA/Protein Kit (Qiagen), according to the manufacturer’s instructions. Polyacrylamide gels were poured in 8 % concentration using 30 % acrylamide/bisacrylamide solution (Bio-Rad), sodium dodecyl sulfate (SDS) (Bio-Rad), ammonium persulfate (APS) (Bio-Rad), tetramethylethylenediamine (TEMED) (Bio-Rad), Resolving Gel Buffer (Bio-Rad) and Stacking Gel Buffer (Bio-Rad). Electrophoresis of protein lysates was performed using 10 µL of each sample, followed by Ponceau S staining. Samples were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). The following antibodies were used for the detection of TET1, TET3, and beta-actin: anti-TET1 in dilution of 1:1000 (Thermo Fisher, PA5-72805), anti-TET3 in dilution 1:1000 (Abcam, ab 139311) and anti-beta-actin in dilution of 1:10 000 (Cell Signaling, 4967L). All membranes were incubated with a secondary anti-rabbit IgG antibody in a dilution of 1:7500, conjugated to horseradish peroxidise (HRP) (Thermo Fisher). Protein signals were detected by chemiluminescence using Clarity Max Western ECL Substrate (Bio-Rad) and quantified with ImageLab software (Bio-Rad). TET1 and beta-actin measurements were normalized to whole-cell lysate content based on the Ponceau S staining.
6
Immunoblotting Analysis of Cellular Proteins
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Whole cell lysates were prepared as described previously [25 (link)]. Equal amounts (30 μg) of total proteins were subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation, followed by immunoblotting analyses with specific antibodies including antibodies against ADAMTS4, p65, p50, p-p65, Glu-1, Hif-1α, Shh, GAPDH and H3 (Abcam, Cambridge, UK). Anti–mouse immunoglobulin G (IgG) and anti–rabbit IgG antibody were purchased from Thermo (Waltham, Massachusetts, USA).
7
Immunofluorescence Labeling of Mitochondrial Proteins
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Cells were fixed with 4% paraformaldehyde, permeabilized in 1% Triton X-100 or 50 µg/ml digitonin for 15 min, diluted in blocking solution (0.1% gelatin in PBS), blocked for 30 min, and stained with the following primary antibodies: an anti-TOMM20 antibody FL-145 (Santa Cruz Biotechnology, 1:2000), an anti-Hsp60 antibody N20 (Santa Cruz Biotechnology, 1:250), or an anti-HA antibody TANA2 (MBL, 1:1000). Among the secondary antibodies used were an Alexa Fluor 488-, 568- or 647-conjugated anti-mouse IgG or an anti-rabbit IgG antibody (Thermo Fisher Scientific, 1:2000). Cells were imaged using a laser-scanning microscope (LSM710 or LSM780; Carl Zeiss). Antibodies used in this study and their Research Resource Identifiers (RRIDs) are listed in Table S1 .
8
DC-SIGN Immunofluorescence Staining Protocol
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Cells were washed three times with ice-cold phosphate-buffered saline (PBS) and fixed with 4% (w/v) paraformaldehyde in PBS for 10 min. After washing three times with PBS, the cells were incubated with 0.5% (v/v) TritonX-100 in PBS for 10 min at room temperature for permeabilization. After washing three times with 0.1% (v/v) TritonX-100 in PBS (PBST), the cells were blocked by incubation with 5% (w/v) BSA in 0.3% (v/v) TritonX-100 in PBS for 1 h at room temperature. After washing three times with PBST, cells were incubated with an anti-DC-SIGN antibody (Cell Signaling Technology, Danvers, MA) and an anti-rabbit IgG antibody labeled with Alexa488 (Thermo Fisher Scientific) sequentially. Counterstaining with Hoechst33342 was also performed, and the resulting cells were mounted with ProLong Diamond Antifade Mountant (Thermo Fisher Scientific). The staining pattern was analyzed by fluorescent microscopy, BZ-X710 (Keyence Corporation, Osaka, Japan).
9
Antibody Selection and Chemical Inhibitors for Studying Retrovirus Release
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Rabbit polyclonal anti-MuLV p30 antiserum was described previously [13 (link)]. Mouse monoclonal anti-HIV-1 p24CA antibody (YDHIVgp24) was purchased from MyBioSource (San Diego, CA, USA). β-Tubulin was used for the loading control in western blots and was detected by rabbit anti-β-Tubulin antibody (Cell Signaling Technology, Danvers, MA, USA). For western blots, we used an anti-mouse IgG antibody conjugated with horseradish peroxidase (Thermo Fisher Scientific, Waltham, MA, USA) and an anti-rabbit IgG antibody conjugated with horseradish peroxidase (Thermo Fisher Scientific, Waltham, MA, USA). The antibodies for LC3 (Cell Signaling Technology) and p62/SQSTM1 (Proteintech Group, Inc., Rosemont, IL, USA) were used to check autophagic flux in the cells expressing Arf6Q67L. To examine the role of Arf6 in MuLV release, the following chemical inhibitors were used: UNC3230 (MedChemExpress, Monmouth Junction, NJ, USA), ML299 (Aobious Inc., Gloucester, MA, USA), FIPI (Tocris, Minneapolis, MN, USA), LY294002, wortmannin, rapamycin, and bafilomycin A1 (Cayman Chemical, Ann Arbor, MI, USA). MG132 and lactacystin were purchased from AdipoGen Life Sciences (San Diego, CA, USA).
10
Immunocytochemical Staining of Neurons
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The neuronal cells (11 DIV) or SH-SY5Y cells growing on glass coverslips were fixed with 10% neutral formaldehyde solution for 15 min at room temperature. The cells were permeabilized with 0.1% Triton X-100 in PBS containing 5% swine serum for 1 h at room temperature and incubated with the primary antibody overnight at 4°C. The cells were then reacted with anti-mouse IgG antibody or anti-rabbit IgG antibody conjugated with Alexa Fluor 488 (Thermo Fisher Scientific) for 1 h at room temperature, and observed under a confocal laser-scanning microscope system, FV-1000D (Olympus, Tokyo, Japan). Under the culture condition, 98% of primary hippocampal neurons were immunoreactive for the neuronal marker MAP2 on double immunocytochemistry with anti-MAP2 antibody and anti-GFAP antibody as the astroglial markers.
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