Western blot analysis of extracts of HeLa, A549 and A431 cells using Keratin 17/19 (D32D9) XP® Rabbit mAb. As expected, keratin 17 is detected in HeLa and A431 cells while keratin 19 is found in A549 cells.
Immunohistochemical analysis of paraffin-embedded HeLa cells (keratin 17 positive) (left), A549 cells (keratin 19 positive) (middle), and Jurkat cells (keratin 17/19 negative) (right) using Keratin 17/19 (D32D9) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Keratin 17/19 (D32D9) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Keratin 17/19 (D32D9) XP® Rabbit mAb.
|REACTIVITY||H M R Mk|
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 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
NOTE: Do not allow slides to dry at any time during this procedure.
For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.
|DETECTION REAGENT/SUBSTRATE COMPATIBILITY|
|SignalStain® Boost IHC Detection Reagent (HRP, Rabbit) #8114||SignalStain® Boost IHC Detection Reagent (AP, Rabbit) #18653|
|SignalStain® DAB Substrate Kit #8059||SignalStain® Vibrant Red Alkaline Phosphatase Substrate Kit #76713|
|SignalStain® Vivid Purple Peroxidase Substrate Kit #96632|
NOTE: Use of detection reagents other than those specified in this protocol may require further optimization of the primary antibody to account for the different sensitivities of the detection reagents.
posted June 2005
revised April 2020
Protocol Id: 303
Keratin 17/19 (D32D9) XP® Rabbit mAb detects endogenous levels of keratin 17 and keratin 19 proteins.Species Reactivity:
Human, Mouse, Rat, Monkey
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to amino acids near the amino terminus of human keratin 17 and human keratin 19.
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 17 is involved in wound healing and cell growth, two processes that require rapid cytoskeletal remodeling (7). Keratinocytes deficient in keratin 17 exhibit abnormal Akt/mTOR signaling and fail to produce an increase in translation, cell size, or growth; these cells also exhibit abnormal 14-3-3σ localization. As 14-3-3σ typically associates with keratin 17, these results imply that Akt/mTOR signaling results in sequestration of 14-3-3σ with keratin 17 in the cytosol, which is required for translation and cell growth. Phosphorylation of keratin 17 on Ser44 may provide a docking site for 14-3-3σ binding (8).
Explore pathways + proteins related to this product.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.