HEK293T cells were transiently transfected with 1 μg of expression constructs for V5/His-tagged full-length (amino acids 1–271) or N-terminally truncated (amino acids 27–271; ΔN-JMJD8) JMJD8. JMJD8 was detected in fixed cells using anti-6×His antibody (Abcam, ab18184) and Alexa Fluor 488–conjugated secondary antibody (Thermo Fisher, a-11001). Images were captured using a Leica SP8x confocal microscope. Nuclear and cytoplasmic fractions were isolated from cells with standard protocols using a Dounce homogenizer. Standard immunoblotting was performed to detect JMJD8 using the anti-6×His antibody (Abcam, ab18184). Antibodies to nuclear lamin A (Santa Cruz Biotechnology, sc-20680) and tubulin (Cell Signaling Technology, 2144) were used to mark the nuclear and cytoplasmic fractions, respectively.
Ab18184
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab18184 is a primary antibody. It is an IgG antibody produced in rabbit and targeted against an unspecified antigen. The antibody is suitable for use in various immunological techniques, including Western blotting and immunohistochemistry. Further details about the specific antigen or recommended applications are not available.
Automatically generated - may contain errors
Lab products found in correlation
Ab205606, by Abcam (4 mentions)
F1804, by Merck Group (3 mentions)
Ab1791, by Abcam (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Ab5392, by Abcam (2 mentions)
Ibright fl1000, by Thermo Fisher Scientific (2 mentions)
Immobilon fl polyvinylidene difluoride pvdf membrane, by Merck Group (2 mentions)
Human anti sars cov 2 spike s ecd rbd, by Thermo Fisher Scientific (2 mentions)
Mouse iggk bp hrp, by Santa Cruz Biotechnology (2 mentions)
Goat anti human hrp, by Thermo Fisher Scientific (2 mentions)
Blotting grade blocker, by Bio-Rad (2 mentions)
Complete protease inhibitor cocktail tablet, by Roche (2 mentions)
Nis elements software, by Nikon (2 mentions)
Ab49923, by Abcam (2 mentions)
Ab32072, by Abcam (2 mentions)
Ab6789, by Abcam (2 mentions)
Ab21623, by Abcam (2 mentions)
Ab6721, by Abcam (2 mentions)
Ab6728, by Abcam (2 mentions)
Anti gm130, by BD (2 mentions)
78 protocols using ab18184
1
Subcellular Localization of JMJD8
2
Transient Protein Expression in 293-F Cells
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Cloning constructs were used to transfect FreeStyle 293-F cells that were grown in suspension using FreeStyle 293 expression medium (Life Technologies) at 37°C in a humidified 8% CO2 incubator rotating at 125 rpm. Cells were grown to a density of 2.5 million cells per ml, transfected using PEI (4 μg/ml in cell suspension) and DNA (1200 ng/ml in cell suspension), and cultivated for 3 days. The supernatants were harvested and proteins purified by His SpinTrap columns according to manufacturer’s instructions (Cytiva, 95056-290). The eluted protein was transferred to phosphate-buffered saline (PBS) via buffer exchange using Amicon Ultra-4 ultrafiltration column with 50 kDa cut-off (Millipore, UFC805008). Protein concentration was determined by His-tag specific ELISA using a mouse anti-His-tag antibody (Abcam, #ab18184) and a goat anti-mouse IgG Fc antibody conjugated to alkaline phosphatase (Southern Biotech, #SBA-1033-04) as detection reagent. Protein production was confirmed by SDS-PAGE and western blot using a mouse anti-His antibody (Abcam, #ab18184) and an IRDye 800CW donkey anti-mouse antibody (Li-Cor Biosciences, #925-32212).
3
APPBP2 and PPM1D Protein Interactions
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To conduct the experiments, four additional plasmids were constructed. Flag, Flag-APPBP2, His, and His-PPM1D were cloned into pCDNA 3·1, respectively. For APPBP2 co-IP in A549 (or H1299) cells, the cells transformed by lipo-3000, expressing Flag-APPBP2, PPM1D, and SPOP, were cultured for 72 h. Meanwhile, the transformed cells expressing Flag, APPBP2, PPM1D, and SPOP were set as the negative control group. For PPM1D co-IP in the A549 (or H1299) cells, the transformed cells expressing His-APPBP2, PPM1D, and SPOP were cultured for 72 h. The transformed cells expressing His, APPBP2, PPM1D, and SPOP were set as the control group. The cells were lysed for co-IP and were incubated with beads (pierce, 6149) which covalently pre-couple Flag antibody (abcam,ab205606) or His antibody (abcam, ab18184). Some cell lysis, which was incubated with the beads covalently pre-coupled with human IgG, was set as another negative control. The protein binding complex was isolated by centrifuging. The precipitates were diluted with SDS sample buffer, separated on a 10% SDS–PAGE gel and subjected to immunoblotting with the corresponding antibodies.
4
Amyloid Precursor Protein and Presenilins Profiling
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The following primary antibodies were used: anti-APP CT (A8717, Sigma-Aldrich, St. Louis, MO); anti-β-amyloid, 17–24 (clone 4G8) (800701, BioLegend, San Diego, CA); anti-PS1 NT raised against the N terminus of PS1 (APS11) (ab15456, Abcam, Cambridge, MA); anti-PS1 CT raised against the C-terminus of PS1 (mAb5643, Cell Signaling Technology, Danvers, MA); anti-PS1 loop raised against the loop domain between transmembrane domains 6 and 7 of PS1 (E2000Y) (ab76083, Abcam, Cambridge, MA); anti-Syt1 (AB5600, Millipore, Temecula, CA); anti-Gapdh (mAb2118, Cell Signaling Technology, Danvers, MA); anti-MAP2 (ab5392, Abcam, Cambridge, MA); anti-His (ab18184, Abcam, Cambridge, MA); anti-V5 (ab9116, Abcam, Cambridge) and anti-FLAG M2 (F1804, Sigma-Aldrich, St. Louis, MO). Alexa Fluor 488 (ThermoScientific, Waltham, MA) and Cy3-labeled corresponding secondary antibodies (Jackson ImmunoResearch, West Grove, PA) were used for confocal microscopy imaging, and IRDye680/800- (Li-COR, Lincoln, NE) or HRP- (Jackson ImmunoResearch, West Grove, PA) conjugated ones were used for western blotting.
5
Western Blot Analysis of Outer Membrane Vesicles
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OMVs were lysed with 1% Triton supplemented with cOmplete Protease Inhibitor Cocktail Tablets (cat# 11697498001, Roche). Samples were mixed with sample buffer with/without dithiothreitol (DTT), heated to 95°C for 10 mins, and subjected to electrophoresis in 4–12% Bis–Tris polyacrylamide gels (Thermo Fisher). Proteins were transferred to Immobilon‐FL polyvinylidene difluoride (PVDF) membranes (Merck Millipore), which were subsequently blocked with 5% blotting grade blocker (cat# 170–6404, BioRad) powder in PBS. Blots were probed with primary antibodies: human anti‐SARS‐CoV‐2 Spike (S‐ECD/RBD) (cat# bcb03, Thermo Fisher, 1:1000, non‐reducing conditions), anti‐Salmonella typhimurium LPS (cat# ab8274, reducing conditions, 1:1000), and mouse anti‐6xHis (ab18184, Abcam, 1:2000, reducing conditions) in 5% blocking buffer in PBS containing 0.1% v/v Tween 20 (PBS‐T), incubating overnight at 4°C on a shaker. Blots were washed 3x with PBS‐T and incubated for 1 h at room temperature with appropriate secondary antibodies: mouse IgGk‐BP‐HRP (cat# sc‐516102, SantaCruz) or goat anti‐human‐HRP (cat# 31410, Thermo Scientific), diluted 1:10,000 in 5% blocking buffer. After washing 3x with PBS‐T and 2x with PBS, SuperSignal West Pico PLUS Chemiluminescent Substrate (cat# 34580, Pierce) was used for detection with an iBright FL1000 (Thermo Fisher) imager in chemiluminescence mode.
6
Immunofluorescence Imaging of Drosophila S2 Cells
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Transfected or infected Drosophila S2 cells were used for immunofluorescence imaging. The transfection and infection procedures were as described in the section above. Cells were harvested and processed for immunofluorescence imaging as previously described (55 (link)). For protein A immunostaining, 1:500 dilution of the rabbit antiserum against protein A was used [Antibody R1194 (26 (link))]. For His-tag immunostaining, 1:100 dilution of mouse monoclonal, clone H8, antibody (ab18184, Abcam) was used. Widefield epifluorescence microscopy was performed on the Nikon Ti microscope and the images were acquired using NIS-Elements software (Nikon). Additional image processing was done using the Fiji (ImageJ) software package (56 (link)). PCC were calculated using the EzColocalization plugin for ImageJ (57 (link)) where, −1 = complete anticolocalization, 0 = noncolocalization, and 1 = complete colocalization.
7
Protein-Protein Interaction Pulldown Assay
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The assay was performed as follows25 (link). Briefly, the purified proteins were used to perform the pull-down assay in a reaction system comprising 800 µL PBS buffer, 5 µM (final concentration) MBP-RpfG and HtsH1C-Flag-His, HtsH2C-HA-His or HtsH3C-Myc-His proteins, and 50 µL Dextrin Sepharose High Performance (Sigma-Aldrich, St. Louis, MO, USA). All samples were incubated at 4 °C overnight. The agarose was collected by centrifugation and washed ten times with PBS containing 1% Triton X-100 to remove non-specifically bound proteins. The MBP-bead-captured proteins were eluted by boiling in 6× SDS loading dye for 10 min. These samples were subjected to SDS-PAGE and Western blotting. Protein detection involved the use of MBP-specific (ab49923), Flag-specific (ab1162), HA-specific (ab187915), Myc-specific (ab32072), and His-specific (ab18184) antibodies obtained from Abcam, UK.
8
Hadal Sea Cucumber Transcriptome Analysis
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Hadal sea cucumber was collected at the depth of 6500 m in the Mariana Trench (10° 57.1693′ N 141° 56.1719′ E). Total RNA was extracted using RNeasy Plus Universal Kits from Qiagen, Hilden, Germany, and reverse-transcribed to cDNA. The transcriptome was obtained by sequencing assembly and annotation by Novogene Company (Tianjin, China). The following reagents were purchased from Takara, Tokyo, Japan: PrimeScriptTM II 1st strand cDNA Synthesis Kit, PrimeSTAR® GXL DNA Polymerase, E. coli DH5α, and pG-KJE8/BL21 competent cells, pCold II vector, restriction enzymes BamH I, and Pst I, T4-DNA ligase, and DNA and protein markers. The 1 mL Ni-NTA affinity column, BCA protein assay kit, primers, and trypsin/chymotrypsin complex (2400:400) were obtained from Sangon Biotech Company, Shanghai, China. Polyvinylidene difluoride (PVDF) membrane was obtained from Millipore Company, USA. The primary (ab18184) and secondary antibodies (ab6789) were obtained from Abcam, Cambridge, UK. Pierce™ ECL Plus Western blot analysis substrate was obtained from ThermoFisher, Waltham, MA, USA.
9
Determination of Oligomeric States of Purified Proteins
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The oligomeric statesof purified 4MtG, hrH1, and hrH3 proteins were determined using the soluble Bis [sulfosuccinimidyl] (BS3) crosslinker (Thermo Scientific, Waltham, MA) in a crosslinking reaction to fix the polymeric structures of proteins followed by reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Supplementary Fig. 5a, b ) and Western blot using anti-His antibody at 1 µg/ml (Cat. No. ab18184, Abcam) (Supplementary Fig. 5c, d ), as described previously32 (link). Briefly, 1 μg recombinant protein was incubated at room temperature in the presence of 4 mM BS3 for 30 min. The crosslinking reaction was stopped by the addition of 1 M Tris-HCl pH 8.0 to a final concentration of 50 mM.
10
Virus Envelope Glycoprotein Binding
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Cells were transfected with VesG and RDpro expression plasmids20 (link), 25 (link) by lipofection using FuGENE6 (Promega, Madison, WI, USA). Cells were plated in U-bottom 96-well plates at equal densities. Cells were then incubated with the extracellular polyclonal VSV-Poly20 (link) or anti-RD114 antiserum (NCI, Rockville, MD, USA) at 1:200 or 1:500 dilution, respectively, or 3 μg/mL sLDLR in 1% BSA in PBS in a total reaction volume of 100 μL. After washing twice with PBS, cells stained with anti-VSVind.G and anti-RDpro antibodies were incubated with their respective secondary antibodies. On the other hand, the cells incubated with sLDLR were stained with an anti-6XHis-tag antibody, ab18184 (Abcam, UK), against the C-terminal 6XHis-tag on sLDLR to probe for sLDLR binding. Cells were then washed twice with PBS, fixed in 2% paraformaldehyde (PFA) in PBS, and analyzed via flow cytometry.
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