Western blot analysis of extracts from various cell lines using YTHDF2 Antibody.
Western blot analysis of extracts from 293T cells, either transfected with non-targeting siRNA (293T siNT) or siRNA targeting YTHDF2 (293T siYTHDF2), Jurkat, and Raji cells using YTHDF2 Antibody (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
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Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised October 2016
Protocol Id: 10
YTHDF2 Antibody recognizes endogenous levels of total YTHDF2 protein.Species Reactivity:
Human, Mouse, Rat, Monkey
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly167 of human YTHDF2 protein. Antibodies are purified by peptide affinity chromatography.
N6-methyladenosine (m6A) is an abundant RNA modification that plays an important role in mRNA splicing, processing, and stability. The m6A modification is specifically recognized by members of the YT521B homology (YTH) domain-containing family (YTHDF), consisting of YTHDF1, YTHDF2, and YTHDF3. All three members of the YTHDF family are primarily cytosolic proteins that share similar sequence and domain structure, including a conserved C-terminal YTH domain that specifically interacts with m6A (1). Despite these similarities, recent studies suggest that YTHDF proteins are involved in distinct regulatory functions with minimal overlap. Specifically, YTHDF1 binding has been reported to promote enhanced mRNA translation, but has no measurable effect on mRNA stability (2). Conversely, YTHDF2 binding appears to promote mRNA degradation, but has minimal effect on translation efficiency (3). The function of YTHDF3 is less clear, but it has been proposed to function as an auxiliary protein for both YTHDF1 and YTHDF2, helping to promote either increased mRNA translation or decay, respectively (4). Additional studies offer a different viewpoint, suggesting that all three YTHDF proteins initiate mRNA degradation (5), or mediate increased mRNA stability and protein expression (6), promoting the idea that these proteins may carry out similar rather than distinct functions.
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