After harvesting MEF WT and WTAP KO cells, they were washed with PBS. Cells were fixed with of 2% Formaldehyde in PBS for 15 min at RT. Subsequently, the cells were permeabilized with 0.5% saponin buffer in PBS. The first antibody was applied for 90 min at RT in 0.5% Saponin buffer in PBS. Along the generated α-m6A antibody clones, the α-m6A antibody from Abcam (ab151230) was applied. After washing in PBS, the cells were incubated for 30 min at RT with the second antibody (Alex647-conjugated goat α-rat IgG, BioLegend, poly4054 or rabbit IgG, Invitrogen) in 0.5% Saponin buffer in PBS. For detection of the biotinylated antibodies, APC-conjugated streptavidin was applied. To stain WTAP, cells were fixed and permeabilized by using the Foxp3/Transcription Factor staining buffer set (eBioscience) according to manufacturer's instructions. Anti-WTAP antibody (Proteintech, 60188) and FITC-conjugated goat antibody α-mouse IgG (BD Biosciences) were applied. After staining, cells were acquired on a FACS Canto II (BD Biosciences) device and samples were analyzed with FlowJo software.
α m6a antibody
Manufactured by Abcam
The α-m6A antibody is a laboratory tool used to detect and analyze the presence of N6-methyladenosine (m6A) modifications in RNA samples. It is a highly specific antibody that recognizes the m6A modification, allowing researchers to study the role of this epigenetic mark in various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Anti wtap antibody, by Proteintech (1 mentions)
Hybond n membrane, by GE Healthcare (1 mentions)
Hrp conjugated secondary antibody, by GE Healthcare (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Sybr gold, by Thermo Fisher Scientific (1 mentions)
Dot blot apparatus, by Bio-Rad (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Hybond, by Cytiva (1 mentions)
2 protocols using α m6a antibody
1
Immunofluorescence Staining for m6A and WTAP
2
Quantification of m6A RNA Modification
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Total RNA was extracted from extracellular parasites with TRIzol (Ambion) as per the manufacturer. An aliquot of the RNA was run on an agarose gel to check for RNA integrity and for the absence of the 28S rRNA band characteristic of host cell contamination. Genomic DNA was isolated from the same aliquot of parasites with DNeasy Blood and Tissue kit (Qiagen) as per the manufacturer. Indicated amounts of nucleic acids were spotted onto Hybond-N+ membrane (GE Healthcare) with a dot blot apparatus (BioRad). The nucleic acid was fixed to the membrane with a Stratagene crosslinker. After the membrane had dried, total nucleic acid stained with SYBR Gold (Invitrogen) for 5 min. Next, the membrane was blocked for 30 min in a 5% fat-free milk solution for 30 minutes followed by a 1 h incubation with α-m6A antibody (Abcam, ab151230, 1:1,000). The m6A mark was detected by chemiluminescence using an HRP-conjugated secondary antibody (GE Healthcare, NA934V, 1:5,000).
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