Western blot analysis of extracts from HeLa cells, untreated or treated with Staurosporine #9953 (1 μM, 3 hr) using Cleaved PARP (Asp214) (D64E10) XP® Rabbit mAb (Biotinylated) primary antibody and Anti-Biotin (D5A7) Rabbit mAb (HRP Conjugate) as secondary detection antibody.
Relative fluorescence of biotinylated and non-biotinylated peptides determined by #5571 Anti-biotin (D5A7) Rabbit mAb (HRP Conjugate) detection using #7003 LumiGLO® Reagent and 20X Peroxide reagents.
Chemiluminescent detection of phosphorylated and non-phosphorylated Met peptides with #4033 Phospho-Met (Tyr1234/1235) (D26) XP® Rabbit mAb (Biotinylated) in conjunction with #5571 Anti-biotin (D5A7) Rabbit mAb (HRP Conjugate).
Anti-biotin (D5A7) Rabbit mAb is conjugated to the carbohydrate groups of horseradish peroxidase (HRP) via its amine groups. This product has been optimized to detect biotinylated primary antibodies. HRP conjugated antibodies do not require incubation with a secondary antibody. *Do not mix this antibody in solution with any Anti-rabbit antibody. Anti-rabbit antibodies will cross react with this antibody and could result in decreased activity of both Anti-rabbit and Anti-biotin (D5A7) Rabbit mAb.
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 2 mg/ml bovine serum albumin (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.
* 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: 11
posted October 2010
protocol id: 1
Protocol Id: 1
Anti-biotin (D5A7) Rabbit mAb (HRP Conjugate) recognizes biotin attached to proteins, peptides, oligonucleotides and solid matrices.Species Reactivity:
All Species Expected
Monoclonal antibody is produced by immunizing animals with a biotinylated protein.
Chemiluminescence systems have emerged as the best all-around method for western blot detection. They eliminate the hazards associated with radioactive materials and toxic chromogenic substrates. The speed and sensitivity of these methods are unequalled by traditional alternatives, and because results are generated on film, it is possible to record and store data permanently. Blots detected with chemiluminescent methods are easily stripped for subsequent reprobing with additional antibodies. HRP-conjugated secondary antibodies are utilized in conjunction with specific chemiluminescent substrates to generate the light signal. HRP conjugates have a very high turnover rate, yielding good sensitivity with short reaction times.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
LumiGLO is a registered trademark of Kirkegaard & Perry Laboratories.