The primary antibodies for western blot were: HA (901501, BioLegend, dilution 1:5000), BRD4 (ab128874, Abcam, dilution 1:2500), α-Tubulin (ab7291, Abcam, dilution 1:10000), Lamin B1 (ab16048, Abcam, dilution 1:2500), Histone H3 (ab18521, Abcam, dilution 1:2500), H3K27ac (8173, Cell Signaling, dilution 1:2500), Pan-acetyl H3 (39140, Active Motif, dilution 1:2500), MYC (sc-40, Santa Cruz, dilution 1:500) and GAPDH (sc-365062, Santa Cruz, dilution 1:10000). The secondary antibodies used were goat anti-mouse HRP (115-035-003, Jackson ImmunoResearch, dilution 1:5000), goat anti-rabbit HRP (111-035-144, Jackson ImmunoResearch, dilution 1:5000). Antibodies used for ChIP-seq and ChIP-qPCR were: BRD4 (ab128874, Abcam) and rabbit polyclonal IgG (sc-66931, Santa Cruz).
Ab18521
Manufactured by Abcam
Sourced in United Kingdom
Ab18521 is a primary antibody used for the detection of the target protein in various experimental applications. This product is intended for research use only.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Whatman, by Cytiva (2 mentions)
Ab97051, by Abcam (2 mentions)
Ab134091, by Abcam (2 mentions)
Pa 66996, by Thermo Fisher Scientific (2 mentions)
H00003104 b01p, by Novus Biologicals (2 mentions)
Ab13579, by Abcam (2 mentions)
Ab63801, by Abcam (2 mentions)
Ab7291, by Abcam (1 mentions)
Goat anti mouse hrp, by Jackson ImmunoResearch (1 mentions)
Goat anti rabbit hrp, by Jackson ImmunoResearch (1 mentions)
Sc66931, by Santa Cruz Biotechnology (1 mentions)
Ab128874, by Abcam (1 mentions)
H3k27ac, by Cell Signaling Technology (1 mentions)
Myc sc 40, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Ab16048, by Abcam (1 mentions)
Ab18251, by Abcam (1 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
23 protocols using ab18521
1
Western Blot and ChIP Antibody Protocol
2
Western Blot Analysis for Protein and Histone Modifications
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Protein concentrations were detected with Pierce™ BCA Protein Assay Kit (Thermo Scientific™, Waltham, MA, USA, 23225). Samples (15–30 µg of whole protein and 2 µg of histone lysate) were loaded on 4–15% SDS-PAGE gel (Mini-PROTEAN TGX™ Precast Gels, Bio-rad, Hercules, CA, USA, #161-0317). Following electrophoresis (100 V and 60 min), separated proteins were transferred onto the PVDF membrane (Bio-Rad, Hercules, CA, USA, #1620177) at 200 mA for 150 min and immunoblotted with primary antibodies (1:1.000 dilution for Cell Signaling Technology (CST, Beverly, MA, USA) Acetyl-α-Tubulin (Lys40) (D20G3) XP® Rabbit mAb 5335, 1:1000 dilution for Abcam (Abcam, Cambridge, UK) Anti-α-Tubulin antibody ab18251, 1:1000 dilution for Abcam Anti-Histone H3 antibody ab18521, 1:1000 dilution for Abcam Anti-Histone H3 dimethyl K4 antibody ab7766, 1:1000 for Cell Signaling Technology Acetyl-Histone H3 (Lys18) Antibody #9675, 1:1000 dilution for Cell Signaling Technology Acetyl-Histone H3 (Lys27) (D5E4) XP® Rabbit mAb #8173, 1:10,000 diluted Anti-LSD1 Antibody ab129195 [EPR6825]) overnight at 4 °C. The next day, the membrane was incubated with HRP-conjugated secondary antibodies (1:5000 dilution for Abcam, ab205718) for 1 h at room temperature. The signal was detected with BIORAD Chemi-Doc XRS+ System. Band intensities were quantified using ImageJ software (NIH, Bethesda, MD, USA).
3
Protein Detection and Histone Extraction
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Protein detection was performed by incubating MCT1 (1:1000; AB90582, Abcam, Cambridge, UK) and β-actin (1:1000; anti-mouse, cat. no. 4967S; Cell Signalling Technology, Milan, Italy) overnight at 4 °C. For histone protein extraction, we used Abcam histone extraction kit (AB113476, Abcam, Cambridge, UK) according to manufacturer’s protocol. For protein detection, rabbit primary H3K18Lac (1:1000; PTM-1406, PTM-biolabs, IL, USA) and H3 (1:1000; AB18521, Abcam, Cambridge, UK) were used. The next day, the membranes were washed three times in PBS for 5 min and incubated with secondary infrared anti-mouse IRDye800CW (1:5000) in PBS/0.5% Tween-20 for 1 h at room temperature. All antibodies were diluted in Odyssey Blocking Buffer. Protein bands intensity was quantified and normalized to β-actin levels [71 (link),72 (link),73 (link),74 (link)].
4
STING Pathway Activation Analysis
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To measure expression level of STING, phosphorylated STING, p62 and LC3II level, THP-1 cells were treated with mock or CBD, followed by 2’3’-cGAMP stimulation and cells were harvested at 0, 0.5, 1, 3 and 6 hours post 2’3’-cGAMP stimulation. Cell lysate were run on SDS-PAGE and analyzed by western blot with the following primary antibodies: anti-STING (clone T3-680), anti-phosphoSTING (Ser366) (clone D7C3S and E9A9K), anti-LC3II (rabbit polyclonal, Abcam ab51520), anti-p62 (clone EPR4844), anti-histone H3 (rabbit polyclonal, Abcam ab18521).
5
Western Blotting of Spliceosomal Proteins
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For western blotting, proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane (Protran, Whatman). The blocked membrane was incubated with antibodies against the following human proteins Smu1 (1:1000; sc-100896—Santa Cruz Biotechnology, USA), Histone H3B (1:5000; ab18521—Abcam, UK), RED (1:1000; Lührmann Laboratory), FBP21 (1:1000; sc-84249 Santa Cruz Biotechnology, USA), Prp38 (1:3000; Lührmann Laboratory)12 (link), MFAP1 (1:1000; Lührmann Laboratory), Snu114 (1:1500; Lührmann Laboratory)54 (link), SF3A2 (1:2000; Lührmann Laboratory) and SF3B1 (1:1000; Lührmann Laboratory)55 (link), Snu66 (1:2000; Lührmann Laboratory)56 (link), phosphorylated SF3B1 (1:1500; Lührmann Laboratory)31 (link), Prp31 (1:1000; Lührmann Laboratory)57 (link), phosphorylated Prp31 (1:2000; Lührmann Laboratory)30 (link). As secondary antibody horseradish-peroxidase-conjugated Goat-anti-rabbit (1:50,000; 111-035-144—Jackson Immunoresearch, USA) or Goat-anti-mouse (1:10,000; 115-035-003—Jackson Immunoresearch, USA) antibodies were used and bound antibody was detected using an ECL detection kit (GE Healthcare).
6
Immunohistochemical Analysis of Fixed Tissues
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In situ hybridization of formalin-fixed tissues and immunohistochemistry of paraformaldehyde-fixed tissues were performed as described [32 (link)]. The primary antibodies used for immunohistochemistry were against calprotectin (MAC 387; ThermoFisher Scientific), CD163 (GHI/61; Invitrogen), cytokeratin (AE1/AE3; Abcam), cleaved GSDMD (EPR20829-408; Abcam), histone H3 (ab18521; Abcam), IFN-1α (MMHA-2; Fisher), IL-1β (6E10; Novus Biologicals), MxA/Mx1 (4812; Novus Biologicals) and influenza A nucleoprotein (DPJY03, BEI Resources). To quantify expression in tissue, TIFF images taken by confocal microscopy were analyzed using Nikon NIS Elements version 4.50.00. Regions of interest were drawn to encapsulate lung tissue and exclude edges of tissue/airways in the tissue where no tissue is present. Microscope slides were imaged non-repetitively for 10 images per tissue section. Stains of interest were then quantified by recording the binary area (recorded as pixels) and averaging the values to generate individual data points per animal, which were averaged for presentation as single data points.
7
Nuclear Protein Fractionation and Western Blot
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Nuclear fraction proteins were separated using 10% SDS-PAGE with Tris-Gly buffer. Next, proteins ranging in size from 25 to 75 kDa were transferred to a 0.45 μm nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) for 1 h at 200 mA. Proteins less than 25 kDa were transferred to a 0.22 μm nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) in a separate sandwich. The 0.22 µm membranes were removed 30 min after the start of the transfer. The quality of protein transfer, hybridization with primary and secondary antibodies, and protein development were assessed as described previously. For primary hybridization, rabbit antibodies (Abcam, Cambridge, UK) HDAC1 (ab7028, 1:3000) and HDAC3 (ab32369, 1:5000) were used. Goat antibodies (Abcam, Cambridge, UK, ab97051, 1:10,000) were used for secondary hybridization. To control protein loading, rabbit antibodies to histone H3 (Abcam, Cambridge, UK, ab18521, 1:3000) were used. All experiments were performed at least three times.
8
Histone H3 PEGylation by hDot1L
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Mononucleosomes (5 pmol) were combined with the catalytic domain of hDot1L (50 pmol) in 20 μl of PEGylation buffer (25 mM Tris-HCl, 150 mM NaCl, 4 mM EDTA, pH 7.8), and incubated at 30 °C for 10 min. To initiate the reaction, maleimide PEG5K (Nanocs) was added from a stock solution (1 mM maleimide PEG5K in PEGylation buffer) to a final concentration of 200 μM. The reaction mixtures were incubated at 30 °C for 15 min. and then quenched on ice through the addition of 1 μl dithiothreitol solution (100 mM dithiothreitol in PEGylation buffer). The samples were separated by SDS-PAGE, transferred to a PVDF membrane and visualized by blotting for H3 (Abcam, ab18521, 1:500 dilution).
9
Shelterin and DNA Damage Markers
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Telomere vesicles were stained with antibodies to Rad51 (1:100; ab63801, Abcam) and TRF2 (1:300; ab13579, Abcam) and POT1 (1:300; PA-66996, ThermoFisher). Primary human T cells were stained with antibodies to sestrin 1 (1:300; ab134091, Abcam). Shelterin expression in primary human APCs was evaluated with antibodies to TRF2 and POT1 as per telomere vesicles. APCs were also stained with TZAP (H00003104-B01P, Novusbio), Caspase 3 (9661T, Cell Signaling) and H3, (ab18521, Abcam).
10
Shelterin and DNA Damage Markers
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