Western blot analysis of extracts from LNCaP cells (PSA positive) and DU 145 cells (PSA negative) using PSA/KLK3 (D6B1) XP® Rabbit mAb (Biotinylated) (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
This Cell Signaling Technology antibody is conjugated to biotin under optimal conditions. The biotinylated antibody is expected to exhibit the same species cross-reactivity as the unconjugated PSA/KLK3 (D6B1) XP® Rabbit mAb #5365.
Supplied in 136 mM NaCl, 2.6 mM KCI, 12 mM sodium phosphate (pH 7.4) dibasic, 2 mg/ml BSA, and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
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.
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.
Do not add Anti-biotin, HRP-linked Antibody for detection of biotinylated protein markers. There is no need. The Streptavidin-HRP will also visualize the biotinylated markers.
* 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: 265
PSA/KLK3 (D6B1) XP® Rabbit mAb (Biotinylated) recognizes endogenous levels of total PSA/KLK3 protein.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Phe165 of human PSA/KLK3 protein.
Kallikrein 3 (KLK3), also known as Prostate Specific Antigen (PSA), is a member of the glandular kallikrein subfamily of serine proteases (1). It is produced by prostate epithelial cells and is secreted into prostatic ducts. Upon cleavage of 7 amino-terminal amino acids (2), it is activated to liquefy semen in the seminal coagulum. Although PSA/KLK3 is produced in healthy individuals, investigators have found abnormally high levels in the blood of men with advanced prostate cancer (2,3).
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