REACTIVITY | SENSITIVITY | MW (kDa) | Isotype |
---|---|---|---|
H Mk | Endogenous | 48 | Mouse IgG1 |
Western blot analysis of extracts from various cell lines using Aurora A (1F8) Mouse mAb.
Learn more about how we get our images.Confocal immunofluorescent analysis of HeLa cells using Aurora A (1F8) Mouse mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Learn more about how we get our images.Flow cytometric analysis of Jurkat cells using Aurora A (1F8) Mouse mAb and propidium iodide (DNA content). Anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4408 was used as a secondary antibody.
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: Loading of prestained molecular weight markers (#13953, 5 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.
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
Achieve higher quality immunofluorescent images using the efficient and cost-effective, pre-made reagents in our #12727 Immunofluorescence Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Recommended Fluorochrome-conjugated Anti-Mouse secondary antibodies:
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.
Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.
NOTE: Formaldehyde is toxic, use only in a fume hood.
NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.
posted November 2006
revised November 2013
Protocol Id: 148
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.
NOTE: Count cells using a hemocytometer or alternative method.
posted June 2005
revised June 2017
Protocol Id: 406
Application | Dilutions |
---|---|
Western Blotting | 1:1000 |
Immunofluorescence (Immunocytochemistry) | 1:800 |
Flow Cytometry | 1:1600 |
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.
Aurora A (1F8) Mouse mAb recognizes endogenous levels of total Aurora A protein.
Human, Monkey
Monoclonal antibody is produced by immunizing animals with a recombinant protein specific to human Aurora A protein.
Aurora A (AIK) is a cell cycle-regulated Ser/Thr protein kinase that is overexpressed in many tumor cell lines (1-3). Phosphorylation of Aurora A at Thr288 within the kinase activation loop results in a significant increase in its activity and may target the protein for proteasomal degradation during mitosis (4). The closely-related kinase Aurora B (AIM1) has been implicated in multiple mitotic events (5), and siRNA silencing of Aurora B expression results in reduced histone H3 phosphorylation, aberrant chromosome alignment/segregation, and altered survivin localization (6).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. Alexa Fluor is a registered trademark of Life Technologies Corporation. DRAQ5 is a registered trademark of Biostatus Limited.
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Product # | Size | Price |
---|---|---|
12100S | 100 µl | $ 260.0 |