Western blot analysis of extracts from MCF7 cells, treated with CIP or TPA (12-O-Tetradecanoylphorbol-13-Acetate) #4174 (200 nM, 10 min), using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb #4370. Protein A (HRP Conjugate) was used for detection.Learn more about how we get our images
Western blot analysis of extracts from NIH/3T3 cells using Akt (pan) (C67E7) Rabbit mAb #4691. Protein A (HRP Conjugate) was used for detection.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.
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
This product should be used in place of the secondary in the western blotting protocol.
Supplied in 136 mM NaCl, 2.6 mM KCl, 12 mM sodium phosphate (pH 7.4) dibasic, 2 mg/ml BSA, and 50% glycerol. Store at –20°C.
This Cell Signaling Technology product is useful for the detection of IgG. Conjugation of horseradish peroxidase (HRP) to protein A is obtained by cross-linking the amino groups on protein A with the carbohydrate groups on HRP.
Protein A exhibits high affinity for subclasses of IgG from many species (including human, rabbit, mouse, rat, and sheep) and therefore can be used for detection and/or purification of antibodies. Due to its vast array of applications, protein A is the method of choice for many preparative and analytical immunological assays (1).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. XP is a registered trademark of Cell Signaling Technology, Inc. Tween is a registered trademark of ICI Americas, Inc.
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