For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 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.
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 June 2016
Protocol Id: 263
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.
RALY Antibody recognizes endogenous levels of total RALY protein.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro163 of human RALY protein. Antibodies are purified by protein A and peptide affinity chromatography.
RALY is a member of the large family of heterogeneous nuclear ribonucleoproteins (hnRNPs). RALY was initially discovered in lethal yellow mice, a condition where heterozygotes display an all yellow coat, obesity, diabetes, and tumors. In this condition, mRNA to the 5’ untranslated region of RALY fuses to the agouti transcript (1,2). RALY binds to U-rich elements in coding and non-coding mRNAs undergoing translation (3). RALY has been implicated certain cancer types, as it can control transcriptional regulation and splicing of E2F1 and PRMT1 mRNAs (4,5).
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