Western blot analysis of extracts from various cell lines using CLIP1/CLIP170 Antibody.
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Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/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 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
CLIP1/CLIP170 Antibody recognizes endogenous levels of total CLIP1/CLIP170 protein.Species Reactivity:
Human, Mouse, Monkey
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human CLIP1/CLIP170 protein. Antibodies are purified by protein A and peptide affinity chromatography.
Microtubules (MTs) are polarized filaments composed of α/β tubulin heterodimers. The slower growing (minus) ends of MTs are located at centrosomes, and the faster growing (plus) ends extend into the cell periphery. Regulation of MT dynamics is important for multiple cellular functions, including cell division, migration, adhesion, membrane trafficking, and polarity (reviewed in 1).
Cytoplasmic linker protein 1/170 (CLIP1/CLIP170) localizes to the plus ends of MTs (2), and binds to the Rac1/cdc42 effector protein IQGAP1. This complex is involved in establishing cell polarity (3).
CLIP1/CLIP170 also facilitates MT-dependent organelle transport (4), and phosphorylation of CLIP1/CLIP170 by PLK1 and CK2 is required for efficient microtubule–kinetochore attachments in mitosis (5).
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