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
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.
RASSF1 (E7T6O) Rabbit mAb recognizes endogenous levels of total RASSF1 protein. The antibody is predicted to detect the following RASSF1 isoforms: 1A, 1D, 1E, 1F, and 1G. It is not predicted to detect isoforms 1B, 1C, or 1H, as these isoforms lack the amino acid sequence corresponding to the antigen.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Arg50 of human RASSF1 protein.
Ras association domain-containing protein 1 (RASSF1) is a member of the RASSF protein family (RASSF1-10), scaffold proteins whose members are unified by the presence of a Ras association (RA) domain that gives them structural similarity to Ras effector proteins (1). The RASSF1 gene is located in a genomic region exhibiting loss of heterozygosity in a more than 90% of small cell lung cancers, and up to 50% of non-small cell lung cancers (2), strongly implicating the RASSF1 gene as a tumor suppressor. Moreover, RASSF1 expression in tumor cells has been shown to be frequently suppressed by promoter hypermethylation, further suggesting a tumor suppressor function (3-5). Although multiple isoforms (RASSF1A-H) have been described, generated by alternative splicing and/or promoter usage, RASSF1A and RASSF1C appear to be the most widely expressed in normal tissues, with RASSF1A expression lost most frequently in tumor cells (6). Notably, RASSF1A has been identified as an effector protein in the Hippo signaling pathway, where it promotes association between MST1/2 and LATS1/2, leading to suppressive phosphorylation of the transcriptional co-activators YAP and TAZ (7).
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