Western blot analysis of extracts from various cell lines using Cyclophilin A (D2Y4M) Rabbit mAb.
Western blot analysis of extract from C2C12 cells, mock transfected (-) or transfected with siRNA against mouse cyclophilin A (+), using Cyclophilin A (D2Y4M) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower)
Supplied in 10mM sodium HEPES (pH 7.5), 150mM NaCl, 100 μg/ml, 50% glycerol and less than 0.02% sodium azide. Store at -20ºC. 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
Cyclophilin A (D2Y4M) Rabbit mAb recognizes endogenous levels of total cyclophilin A protein.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly18 of mouse cyclophilin A protein.
Cyclophilins are a highly conserved family of peptidylprolyl cis-trans-isomerases (PPIA) that are targets of the immunosuppressant drug cyclosporin A (CsA) (1,2). The complex of cyclophilin and CsA can bind to and inhibit calcineurin which leads to inhibition of the transcription factor NFAT and decreased production of cytokines (3,4). As isomerases, cyclophilins have been proposed to aid in protein folding. Cyclophilin A can bind to the p55 Gag protein of HIV and appears necessary for HIV infection (5,6). There is also some evidence that cyclophilins have nuclease activity and play a role in apoptosis (7).
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