For immunoblot analysis, 20–50 μg of cell lysate was loaded per lane for SDS-PAGE and transferred to PVDF membranes. For immunofluorescence staining, tissue sections (7 μm thick) were fixed using either 50% acetone and 50% methanol, or 4% formaldehyde. Primary antibodies were incubated at 4°C overnight and secondary antibodies were incubated at room temperature for 1 hour. Images were taken using a Zeiss Observer Z1 fluorescence microscope, and the staining signals were quantified using Image J software. The antibodies used in this study for western blotting and immunostaining include: pAb-anti-PRMT1 (Millipore), ms-anti-PRMT1 (Santa Cruz), anti-CSNK1a1 (Santa Cruz), anti-Krt1 (Covance), anti-Krt10 (Neomarkers), anti-Loricrin (Covance), ms-anti-CollagenVII (Millipore), pAb-anti-CollagenVII (Calbiochem), GST (Cell Signaling), phosphoserine pAb (Invitrogen), phospho-threonine Ab (Invitrogen), Anti-phospho Histone H3 (Ser10) pAb (Millipore), Anti 53BP1 pAb (Novus), ASYM24 (Millipore).
Asym24
Manufactured by Merck Group
Sourced in United States
ASYM24 is a piece of laboratory equipment manufactured by Merck Group. It is designed for use in scientific research and analysis. The core function of ASYM24 is to perform automated sample processing and analysis tasks. Further details on its intended use or specific capabilities are not available.
Automatically generated - may contain errors
Lab products found in correlation
Observer z1, by Zeiss (1 mentions)
Anti krt1, by Fortrea (1 mentions)
Anti loricrin, by Fortrea (1 mentions)
Gapdh, by Abcam (1 mentions)
Sym10, by Merck Group (1 mentions)
Lamin b1, by Abcam (1 mentions)
Hdac1, by Abcam (1 mentions)
Ddx17, by Abcam (1 mentions)
Ha 11, by BioLegend (1 mentions)
Lamin a c, by Abcam (1 mentions)
Taf15, by Fortis Life Sciences (1 mentions)
Prmt1, by Abcam (1 mentions)
Dgcr8, by Abcam (1 mentions)
Mono methyl arginine r gg d5a12, by Cell Signaling Technology (1 mentions)
Drosha, by Santa Cruz Biotechnology (1 mentions)
H4r3me2a, by Active Motif (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
Celltiter gloviability kit, by Promega (1 mentions)
Lsm 510 meta confocal microscope, by Zeiss (1 mentions)
Vectashield mounting media with dapi, by Vector Laboratories (1 mentions)
6 protocols using asym24
1
Immunoblot and Immunofluorescence Techniques
2
Protein Immunoprecipitation and Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following antibodies were used for immuno-precipitation and Western Blot experiments, according to the manufacturer's instruction: DGCR8 (Abcam ab90579), EWSR1 (Abcam ab54708), FUS (Bethyl A300–293A), DDX5 (Abcam ab126730), Drosha (Santa Cruz sc-33778), Vinculin (VCL) (Millipore 06–866), PRMT1 (Abcam ab73246), ASYM24 (Millipore), SYM10 (Millipore), Mono-Methyl Arginine (R*GG) (D5A12) (Cell Signaling Technology 8711), LAMIN A/C (sc-6215), Lamin B1 (Abcam ab16048), GAPDH (Abcam ab9484), H3 (Abcam ab1791), H4 (Abcam ab7311), H4R3me2a (Active Motif 39705), TAF15 (Bethyl Laboratories A300–308A), ILF3 (Bethyl A303–615A), ILF2 (sc-271718), DDX17 (sc-130650), HDAC1 (Abcam ab7028) and HA.11 (Biolegend 901513).
MS023 and MS094 compounds were kindly provided by the SGC Toronto—Structural Genomic Consortium (http://www.thesgc.org/scientists/groups/toronto ). Compounds were dissolved in DMSO and used at a final concentration of 10 μM for the indicated time intervals.
MS023 and MS094 compounds were kindly provided by the SGC Toronto—Structural Genomic Consortium (
3
Cell Viability and Arginine Methylation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Basically, the experiments
were performed as reported.41 (link) Briefly,
the cell viability was measured by CellTiter-Glo viability kit (Promega,
Madison WI). 1000 cells were seeded in individual wells of 96-well
plates, with 100 μL culture volume per well. All these leukemia
cell lines were grown in RPMI medium plus 10% fetal bovine serum.
Indicated concentrations of compound50 or the same amount
of DMSO were added to the culture. At 0, 24, and 48 h after the drug
treatment, 100 μL of CellTiter-Glo reagent was added to each
well. Luminescence signals, which are proportional to viable cell
numbers in each well, were measured by microplate reader (Biotek,
Winooski, VT). For detection of total ADMA level in cells, 4 ×
105 cells were cultured with indicated concentrations of50 or DMSO. Cells extracts were harvested after 24 h of treatment
and resolved by SDS–PAGE. Total protein arginine methylation
level was determined by Western blotting of Asym 24 (Millipore) and
D6H8 (Cell Signaling, Beverly, MA) antibodies.
were performed as reported.41 (link) Briefly,
the cell viability was measured by CellTiter-Glo viability kit (Promega,
Madison WI). 1000 cells were seeded in individual wells of 96-well
plates, with 100 μL culture volume per well. All these leukemia
cell lines were grown in RPMI medium plus 10% fetal bovine serum.
Indicated concentrations of compound
of DMSO were added to the culture. At 0, 24, and 48 h after the drug
treatment, 100 μL of CellTiter-Glo reagent was added to each
well. Luminescence signals, which are proportional to viable cell
numbers in each well, were measured by microplate reader (Biotek,
Winooski, VT). For detection of total ADMA level in cells, 4 ×
105 cells were cultured with indicated concentrations of
and resolved by SDS–PAGE. Total protein arginine methylation
level was determined by Western blotting of Asym 24 (Millipore) and
D6H8 (Cell Signaling, Beverly, MA) antibodies.
4
Quantifying aDMA in GFP-expressing Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK-293 T cells were fixed with 4% paraformaldehyde in PBS for 15 min, and then permeabilized with PBS-0.5% Triton X-100 for 10 min. To examine aDMA, cells were blocked with 5% nonfat milk for 1 h at room temperature, and then incubated overnight at 4 °C with rabbit polyclonal anti-aDMA antibody (ASYM24, Millipore). After washing, cells were incubated with the corresponding Alexa Fluor 568-conjugated goat anti-rabbit secondary antibody (1:500, Molecular Probes) at room temperature for 2 h. After washing, they were mounted with Vectashield Mounting Media with DAPI (Vector Laboratories). Images were obtained on a Zeiss LSM 510 META confocal microscope. To quantify the number of cells positive for GFP and aDMA, microscopic fields were randomly selected at 40X magnification. For each field, the number of GFP-positive cells and aDMA-positive aggregates, as well as aggregates with double staining, were manually counted, blinded to condition. These counts were used to calculate the average percentage of aDMA aggregates in cells expressing GFP, GFP-(GR)50 or GFP-(GR)100.
5
Quantitative Analysis of Protein Methylation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The proteins resolved by 1-dimensional (1D) or 2D PAGE were transferred onto a PVDF membrane (Millipore, Billerica, MA, USA). The membranes were incubated overnight at 4°C with antibodies against PRMT1 (1:1,000 dilution, Cell Signaling Technology, Danvers, MA, USA), PRMT4 (1:5,000, Novus Biology, Centennial, CO, USA), PRMT6 (1:1,000 dilution, Cell Signaling Technology), ASYM24 (1:5,000, Millipore), and β-actin (1:10,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). After extensive washing with Tris-buffered saline (TBS), the immunoblot was incubated with peroxidase-conjugated secondary antibody for 1 hour at room temperature and then washed with TBS/0.05% Tween-20 followed by water. The bound antibodies were visualized by the immunoblotting detection reagents (Millipore). The relative intensities of specific signals were quantified using the Kodak MI imaging system (Kodak, Rochester, NY, USA).
6
Antibody Characterization for Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following antibodies were used: anti-ARF4 (11673-1-AP, Proteintech, Chicago, IL); anti-asymmetrical dimethylated arginine (ADMA, #13522, Cell Signaling Technology, Beverly, MA; ASYM24, 07-414, Millipore, Billerica, MA); anti-Bip/GRP78 (BD Bioscience, Franklin Lakes, NJ); anti-Calnexin (10427-2-AP, Proteintech); anti-ERGIC53 (sc-398777, Santa Cruz Biotechnology, Santa Cruz, CA); anti-Flag (F1804, Sigma-Aldrich, St. Louis, MO); anti-GAPDH (#5174, Cell Signaling Technology); anti-green fluorescent protein (GFP) (sc-9996, Santa Cruz Biotechnology); anti-GM130 (BD Bioscience); anti-γ2-COP (sc-14165, Santa Cruz Biotechnology); anti-glutathione-S-transferase (GST) (ab9085, Abcam, Cambridge, UK); nonimmune immunoglobulin (30000-0-AP, Proteintech); anti-PRMT1 (#2449, Cell Signaling Technology); anti-SCYL1 (HPA015015, Atlas, Bromma, Sweden; ab95074, Abcam) and Tau-1 (#MAB3420, Millipore) antibodies. Secondary antibodies HRP-conjugated anti-IgG, Alexa Fluor 488, 568, and 595 were purchased from Abcam, Cell Signaling Technology, Invitrogen, and Proteintech. Arginine methylation inhibitor (oxidized adenosine [AdOx]) and PRMT1 inhibitor (AMI-1) were purchased from Sigma-Aldrich.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!