REACTIVITY | SENSITIVITY | MW (kDa) | Isotype |
---|---|---|---|
H M R | Endogenous | 90-100 | Rabbit IgG |
Western blot analysis of extracts from HCT 116 and HCT 116 Alix knockout (-/-) cells using Alix (E6P9B) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Learn more about how we get our images.Western blot analysis of extracts from various cell lines using Alix (E6P9B) Rabbit mAb.
Learn more about how we get our images.Immunoprecipitation of Alix from K-562 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control, and lane 3 is Alix (E6P9B) Rabbit mAb. Western blot was performed using Alix (E6P9B) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was using as a secondary antibody.
Learn more about how we get our images.Confocal immunofluorescent analysis of HCT 116 cells (left, positive) and HCT 116 Alix knockout (-/-) cells (right, negative) using Alix (E6P9B) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
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: Loading of prestained molecular weight markers (#13953, 10 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.
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: 263
This protocol is intended for immunoprecipitation of native proteins utilizing Protein A agarose beads for analysis by western immunoblot or kinase activity.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
10X Cell Lysis Buffer: (#9803) To prepare 10 ml of 1X cell lysis buffer, add 1 ml cell lysis buffer to 9 ml dH2O, mix.
NOTE: Add 1 mM PMSF (#8553) immediately prior to use.
Proceed to one of the following specific set of steps.
NOTE: To minimize masking caused by denatured IgG heavy chains (~50 kDa), we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (L57A3) mAb (#3677) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127). To minimize masking caused by denatured IgG light chains (~25 kDa), we recommend using Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127).
posted December 2008
revised April 2018
Protocol Id: 409
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:
NOTE: Cells should be grown, treated, fixed and stained directly in multiwell plates, chamber slides or on coverslips.
NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.
posted November 2006
revised December 2010
Protocol Id: 32
Application | Dilutions |
---|---|
Western Blotting | 1:1000 |
Immunoprecipitation | 1:50 |
Immunofluorescence (Immunocytochemistry) | 1:800 |
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.
Alix (E6P9B) Rabbit mAb recognizes endogenous levels of total Alix protein.
Human, Mouse, Rat
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro346 of human Alix protein.
Alix, a phylogenetically conserved cytosolic scaffold protein, contains an N-terminal Bro1 domain, a coiled-coil region and a C-terminal proline-rich domain (1,2). Originally identified as an ALG-2 (apoptosis-linked gene 2)-interacting protein involved in programmed cell death (3,4), Alix also regulates many other cellular processes, such as endocytic membrane trafficking and cell adhesion through interactions with ESCRT (endosomal sorting complex required for transport) proteins, endophilins, and CIN85 (Cbl-interacting protein of 85 kDa) (5,6).
Alix has also been shown to be involved in the biogenesis of exosomes, small secreted vesicles that contribute to cell signaling (7, 8). In addition, Alix interacts with Atg12-Atg3 to promote autophagy and endosomal fusion (9)
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. XP is a registered trademark of Cell Signaling Technology, Inc. DRAQ5 is a registered trademark of Biostatus Limited. Tween is a registered trademark of ICI Americas, Inc.
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Product # | Size | Price |
---|---|---|
92880S | 100 µl | $ 260.0 |