Western blot analysis of extracts from of HT-29 cells, untreated (-), treated with Anisomycin #2222 (25 μg/ml, 30 min; +), or treated with Anisomycin and Calf Intestinal Phosphatase (CIP)/λ phosphatase (+), using Phospho-Keratin 20 (Ser13) (D9M6O) Rabbit mAb (upper), Keratin 20 (D9Z1Z) XP® Rabbit mAb #13063 (middle) or β-Actin (D6A8) Rabbit mAb #8547 (lower).
Confocal immunofluorescent analysis of HT-29 cells, untreated (left), treated with Anisomycin #2222 (25 μg/mL, 30 minutes; center), or treated with anisomycin followed by λ phosphatase (right), using Phospho-Keratin 20 (Ser13) (D9M6O) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
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.
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 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 June 2020
Protocol Id: 10
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.
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 December 2010
Protocol Id: 3
Phospho-Keratin 20 (Ser13) (D9M6O) Rabbit mAb recognizes endogenous levels of keratin 20 protein only when phosphorylated at Ser13.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser13 of human keratin 20 protein.
Keratins (cytokeratins) are intermediate filament proteins that are mainly expressed in epithelial cells. Keratin heterodimers composed of an acidic keratin (or type I keratin, keratins 9 to 23) and a basic keratin (or type II keratin, keratins 1 to 8) assemble to form filaments (1,2). Keratin isoforms demonstrate tissue- and differentiation-specific profiles that make them useful as research biomarkers (1). Research studies have shown that mutations in keratin genes are associated with skin disorders, liver and pancreatic diseases, and inflammatory intestinal diseases (3-6).
Keratin 20 is primarily expressed in gastric and intestinal epithelium, urothelium, and Merkel cells (7). Research studies have shown that keratin 20 is an important marker of colon, liver, pancreatic, Merkel cell, and gastric cancer (8). Serine 13 of keratin 20 is phosphorylated in response to stress in intestinal epithelia, likely through the p38 MAPK pathway (9, 10).
Explore pathways + proteins related to this product.
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