Western analysis of extracts from Hela cells that were untreated, treated with doxorubicin (0.5 µM, 24 hours), or with nocodazole (50 ng/ml, 24 hours), using Cyclin A2 (BF683) Mouse mAb.Learn more about how we get our images.
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.
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 June 2016
Protocol Id: 19
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.
Cyclin A2 (BF683) Mouse mAb detects endogenous levels of cyclin A2 and does not cross-react with other cyclins or Cyclin A1.
Monoclonal antibody is produced by immunizing animals with a recombinant human cyclin A2 protein.
While overcoming the G1/S checkpoint to commence DNA replication requires cyclin E, and traversing the G2/M checkpoint to initiate mitosis requires cyclin B to be present, cyclin A seems to be required for both S-phase and M-phase (1). A number of studies have described the ability of over-expressed cyclin A to accellerate the G1 to S transition causing DNA replication, and cyclin A antisense DNA can prevent DNA replication (2-4). Cyclin A availability is apparently the rate-limiting step for entry into mitosis, and cyclin A is required for completion of prophase (5). At late prophase, cyclin A may no longer be necessary as cdc2/cyclinB1 becomes active (5).
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|4656T||20 µl (2 western blots)||$ 109.0|
|4656S||100 µl (20 western blots)||$ 255.0|