Western blot analysis of extracts from various cell lines using FKPB1A/FKBP12 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
FKBP1A/FKBP12 Antibody recognizes endogenous levels of total FKBP1A/FKBP12 protein.Species Reactivity:
Human, Mouse, Rat
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Phe49 of human FKBP1A/FKBP12 protein. Antibodies are purified by protein A and peptide affinity chromatography.
FKBP12 (FKBP1A) is a prototypical member of the FKBP (FK506 binding protein) family of immunophilins, so named because of the ability of FKBP12 to bind to the immunosuppressive drug FK506 (tacrolimus) (1). The protein is the smallest member in the family and contains only one peptidylprolyl isomerase (PPIase) core domain (FK domain) responsible for its PPIase activity, and its binding to FK506 and other compounds (e.g., rapamycin). When bound to FK506 or rapamycin, the protein:drug complex further binds and inhibits two important signaling molecules: calcineurin, a key enzyme in T cell activation; and the metabolic enzyme mTOR. The inhibition of these pathways has been functionally linked to immunosuppression (1,2). Through its binding properties, FKBP12 also plays regulatory roles in other pathways. For example, the ryanodine receptor (RyR), a type of Ca2+ release channel, exhibits leakiness in the absence of FKBP12 binding, leading to reduced muscle contractility (3). FKBP12 can also bind TGFBR1 and prevent ligand independent activation (4). The protein also mediates MDM2 degradation by binding and disrupting MDM2/MDM4 association, thereby inducing MDM2 self-ubiquitination and enhancing the sensitivity of cells to chemotherapy induced cellular apoptosis (5).
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