Tissue homogenates and crude membranes were prepared as previously described [15 (link), 16 (link)]. SDS-PAGE and immunoblotting was performed as previously described [15 (link)]. GPR37L1 was detected using goat anti-GPR37L1 (C-12) antibody (1:1000; sc-164532, Santa Cruz Biotechnology, USA) that was previously confirmed to be GPR37L1-specific using knockout tissue [15 (link)] and then rabbit anti-goat antibody (1:7500; 61-1620, Invitrogen). β-galactosidase was detected using rabbit anti-β-galactosidase antibody (1:5000; A-11132; Invitrogen) and then donkey anti-rabbit antibody (1:15000, NA934, GE Healthcare, Australia). GAPDH was detected using rabbit anti-GAPDH antibody (1:10000; 14C10, Cell Signaling, USA) and then donkey anti-rabbit antibody (1:20000, NA934, GE Healthcare). SuperSignal™ West Pico (34080, Invitrogen) or Clarity™ (1705060, Bio-Rad, USA) were used as chemiluminescent substrates. Densitometry was performed using ImageJ software (https://imagej.nih.gov/ij/ ) on non-saturated chemiluminescent exposures. Pixel density of background was measured and subtracted from subsequent measurements taken on the same image. Pixel density of GPR37L1 was measured in reference to the predominant, cleaved receptor species [15 (link)] and adjusted to equivalent protein abundance using each sample’s own GAPDH pixel density score.
Na934
Manufactured by GE Healthcare
Sourced in United Kingdom, United States
The NA934 is a laboratory instrument designed for the analysis and measurement of various samples. It is capable of performing a range of analytical functions, but a detailed description cannot be provided while maintaining an unbiased and factual approach without extrapolation on the intended use.
Automatically generated - may contain errors
Lab products found in correlation
Na931, by GE Healthcare (60 mentions)
Nxa931, by GE Healthcare (7 mentions)
Pvdf membrane, by Merck Group (6 mentions)
Ecl plus, by GE Healthcare (6 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (5 mentions)
Na935, by GE Healthcare (5 mentions)
Hybond, by Cytiva (4 mentions)
A10042, by Thermo Fisher Scientific (4 mentions)
Na931v, by GE Healthcare (4 mentions)
Ab1791, by Abcam (4 mentions)
Nitrocellulose membrane, by Bio-Rad (3 mentions)
Phosstop, by Roche (3 mentions)
T6074, by Merck Group (3 mentions)
Image lab software, by Bio-Rad (3 mentions)
Np0005, by Thermo Fisher Scientific (3 mentions)
Pierce ecl, by Thermo Fisher Scientific (3 mentions)
Np0007, by Thermo Fisher Scientific (3 mentions)
Imagequant tl, by Cytiva (3 mentions)
Ab4729, by Abcam (3 mentions)
Ab3280, by Abcam (3 mentions)
129 protocols using na934
1
GPR37L1 Protein Detection and Quantification
2
Western Blot Analysis of Protein Extracts
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Cells were grown in a 12-well plate at a density of 2 × 105 cells/well for 48 h. Cells were washed with PBS and lysed in lysis-buffer containing RIPA 1X (Sigma-Aldrich), anti-phosphatase and anti-protease (Roche). Protein concentration from total protein extracts was quantified using the BCA Protein Assay kit (Thermo Fisher Scientific). 20 μg of protein for each sample was boiled for 5 min in NuPage buffer (Invitrogen) with reducing agent (Invitrogen) and RIPA 1X. Samples were then subjected to SDS-PAGE and transferred onto PVDF membranes (Millipore). Membranes were activated by absolute ethanol followed by electro-transfer and saturation with PBS supplemented with 0.1% Tween 20 (Sigma-Aldrich) and 5% nonfat milk. Membranes were incubated with primary antibody, washed three times in PBS supplemented with 0.1% Tween 20 (Sigma-Aldrich), incubated with mouse (NA931; GE Healthcare) or rabbit (NA934; GE Healthcare) secondary antibody coupled to peroxidase, washed three times in PBS supplemented with 0.1% Tween 20 (Sigma-Aldrich), and revealed by chemiluminescence with ECL Western blotting detection system (GE Healthcare). Images were acquired with the PXi4 GeneSys software version 1.3.9.0.
3
Hippocampal Protein Expression Analysis
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The hippocampus was removed and homogenized in RIPA buffer with protease inhibitor cocktail (Nacalai tesque). The homogenate was centrifuged at 15,000 rpm for 10 min at 4 °C. The supernatants were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (e-PAGEL-HR, ATTO), and proteins transferred onto an Immobilon-P membrane (Clear Blot Membrane-P plus, ATTO). The membranes were blotted with antibodies of anti-Iba-1 (019–19,741, rabbit, 1:500, Wako), anti-synaptophysin (ab32127, rabbit, 1:1000, Abcam), anti-PSD95 (ab18258, rabbit, 1:10,000, Abcam) and anti-β-actin (G043, mouse, 1:1000, Abm) antibodies. The sections were treated with secondary antibody (NA931 and NA934, 1:10,000, GE Healthcare) and protein bands visualized with a chemiluminescence detection system (ECL Select Western Blotting Detection Reagent, Amersham). Immunoblot signals were analyzed by a LAS-4000 digital imaging system (Fujifilm, Tokyo, Japan). Band intensities were quantified and divided by their corresponding loading controls (β-actin) (n = 3 animals per group).
4
Flow Cytometry, ELISA, and Western Blot Analysis
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For flow cytometry, cells were washed in PBS supplemented with 1% FCS, 1% Sodium Azide and 3 mM EDTA (FACS buffer) and stained with a cocktail of anti-CD3 PE, anti-CD4 APC-Cy7 and anti-CD25 FITC (eBioscience). Cells were then fixed and permeabilized using eBioscience FoxP3 staining kit (77-5775-40) and stained with anti-Foxp3 APC. Samples were examined using a BD LSRFortessa flow cytometer and analyzed using FlowJo software (Treestar). For ELISA, supernatants from cell culture were removed and examined for their cytokine content using ELISA kits for IL10 (DY417) and IFNγ (DY485) according to the manufacturer's instructions (R&D systems). For Western blotting, cerebellar slices were scraped from the culture membrane and suspended in PTxE buffer (PBS, 1% Triton-x, 1 mM EDTA) using mechanical homogenisation and sonication. Western blotting was performed on tissue suspensions as we have described previously [30] (link). Primary antibodies used were: anti-MOG (Millipore, MAB5680), anti-myelin basic protein (MBP) (Abcam ab40390), anti-actin (ECM Biosciences, AM2021), anti-neurofilament H (NFH) (1/1000 dilution: Millipore, MAB5539). Secondary antibodies used were: HRP conjugated mouse (Sigma, A8924), and HRP conjugated rabbit (GE Healthcare, NA934).
5
Immunoblotting Technique for Protein Analysis
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Samples were solubilized in sample buffer (3X) and boiled at 95 °C for 5 min for immunoblotting. All samples were electrophoresed on 10% (w/v) sodium dodecyl sulphate-polyacrylamide (SDS-PAGE) gels. Separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes. For all immunoblot experiments, membranes were blocked for 1 h at ambient temperature in blocking buffer composed of 5% (w/v) skim milk powder in TBS. PVDF membranes were incubated overnight at 4 °C with primary antibodies. A complete list of primary antibodies and the appropriate methodology used is shown in Additional file 1 : Table S1. Membranes were then washed with TBS containing 0.05% Tween 20 and incubated for 1 h at ambient temperature with the following secondary antibodies diluted in blocking buffer: either α-mouse horseradish peroxidase (HRP)-conjugated (NA931, GE Healthcare; 1:5000), α-rabbit HRP-conjugated (NA934, GE Healthcare, 1:5000), or α-goat HRP-conjugated (sc-2433, Santa Cruz, 1:5000) antibody. Visualization of immunoblot labelling was achieved using chemiluminescence with the Western Lighting Plus ECL kit (Perkin Elmer).
6
Western Blot Protocol for DDR Proteins
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Western blots were probed with anti-WDR48 (1:1000, PA5–24007, Thermo Fisher), anti-RAD51AP1 (1:500, ab88370, Abcam), anti-USP1(1:500, ab108104, Abcam), anti-FANCD2 (1:1000, sc-20022, FI17, Santa Cruz Biotechnology), or anti-Ku86 (1:5000, sc-515736, B-4, Santa Cruz Biotechnology) antibody. After incubation with the secondary antibody (1:5000, NA934, NXA931, GE Healthcare), blots were developed using the SuperSignal ECL substrate (34580, Thermo Fisher).
7
Immunohistochemical Profiling of Liver Tissues
8
Immunoblotting Antibody Detection Protocol
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Following antibodies were used to specifically detect proteins in immunoblotting: AR (sc-816), IFI16 (sc-8023), ASC (sc-22514), IL-1β (sc-7884), and IL-18 (sc-7954) from Santa Cruz Biotech (Santa Cruz, CA, USA); Caspase-1 (AHZ0082) from Invitrogen (Grand Island, NY, USA); Anti-STAT1 (cat # 9172), p-STAT1 (cat # 9171), and β-actin (cat # 4967) from Cell Signaling Technology (Danvers, MA, USA). Rabbit polyclonal antibodies that we raised against the C-terminal AIM2 peptide that specifically detected two human hAIM2 isoforms have been described [31 (link)]. Specific custom anti-peptide rabbit polyclonal antibodies were raised against a peptide (REEQETGICGSPSSARSV) in the POP3 protein, which detected an IFN-inducible POP3 protein of an expected size (~18 kDa) in total cell extracts from IFN-treated THP-1 cells as described [15 (link)]. Horseradish peroxidase (HRP) conjugated secondary anti-mouse (NXA-931) and anti-rabbit (NA-934) antibodies were from GE Healthcare Biosciences (Piscataway, NJ, USA).
9
Western Blot Analysis of Extracellular Vesicle Markers
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Protein samples were loaded onto 4–12% Tris-glycine gels. After electorophoresis and transferring to nitro-cellulose membranes, the membranes were blocked in Tris-buffered saline containing 5% no-fat milk for 60 min at room temperature. Membranes were then incubated overnight at 4 °C with anti-Ascl1 antibody (1:100, Santa Cruz Biotechnology, sc-374104), anti-CD63 antibody (1:200, Santa Cruz Biotechnology, sc-5275), anti-Alix antibody (1:1000, Cell Signaling Technology, 2171), anti-TSG101 antibody (1:200, Santa Cruz Biotechnology, sc-7964), anti-ApoA1 antibody (1:200, Santa Cruz Biotechnology, sc-69755), anti-HRS antibody (1:1000, Santa Cruz Biotechnology, sc-271455), anti-Occludin antibody (1:500, Invitrogen, 71-1500), anti-Claudin-5 antibody (1:1000, Invitrogen, 35-2500) and anti-β-actin antibody (1:2000, Sigma-Aldrich, A5441). After incubation with peroxidase-conjugated secondary antibodies, visualization was enhanced by chemiluminescence (GE Healthcare, NA931- anti-mouse, or NA934- anti-rabbit). Optical density was assessed using the ImageJ. Uncropped versions of the blots are shown in the Source Data.
10
Yeast Protein Extraction for Immunoblotting
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Yeast protein extracts were prepared as previously described [52 (link)]. In brief, cells in early-log phase were harvested at 5000×g for 10 min at 4°C. The cell pellet was washed twice in cold water and resuspended in 0.1 M NaOH. Following 5 min alkaline treatment at room temperature, the cells were centrifuged at 10000×g for 1 min and the supernatant was discarded. The cell pellets were resuspended in Laemmli SDS sample buffer (Alfa Aesar), incubated for 5 min at 95°C and centrifuged at 10000×g for 30 s to remove cell debris; 0.50 mg of wet cell weight was analyzed by immunoblotting. Primary antibodies were rabbit anti-HA-tag (Abcam, ab9110, 1:3000), rabbit anti-NAA30 (Sigma–Aldrich, HPA057824, 1:1000), rabbit anti-NAA60 (Sigma–Aldrich, SAB1102546, 1:1000) and mouse anti-actin (Abcam, ab8224, 1:5000). Secondary antibodies were HRP-conjugated anti-rabbit and anti-mouse (both from GE Healthcare, NA934 and NA931, 1:5000).
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