Western blot analysis of extracts from various cell lines using hnRNP C1/C2 (D6S3N) Rabbit mAb.
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
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
hnRNP C1/C2 (D6S3N) Rabbit mAb recognizes endogenous levels of total hnRNP C1/C2 protein.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ile217 of human hnRNP C1/C2 protein.
Heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNP C1/C2) has multiple biological functions including transcriptional regulation, DNA repair, and RNA processing. hnRNP C1/C2 acts as a ‘molecular ruler’ in the mRNA processing pathway, committing nascent transcripts from the chromatin template to the mRNA export pathway once the nascent transcript becomes longer than 200-300 nucleotides (1). hnRNP C1/C2 associates with SWI/SNF and NurD family members to form the locus control region (LCR)-associated remodeling complex (LARC), which binds to β-globin gene promoter to prevent transcriptional silencing. Studies indicate that without hnRNP C1/C2, LARC does not associate with its target DNA sequence (2,3). hnRNP C1/C2 and other hnRNP family members interact with DNA damage response (DDR) proteins (4). hnRNP proteins regulate double stranded break (DSB) repair by promoting either homologous recombination (HR) or non-homologous end joining (NHEJ) (4). hnRNP C1/C2 downregulates the expression of miR-21, which leads to the increased expression of programmed cell death 4 (PDCD4) protein in glioblastoma multiforme (GBM) (5). Research studies have shown that silencing of hnRNP C1/C2 renders GBM cells more susceptible to apoptosis (5).
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