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92880
Alix (E6P9B) Rabbit mAb
Primary Antibodies

Alix (E6P9B) Rabbit mAb #92880

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APPLICATIONS

REACTIVITY SENSITIVITY MW (kDa) Isotype
H M R Endogenous 90-100 Rabbit IgG
Western Blotting
Western Blotting

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).

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Western Blotting
Western Blotting

Western blot analysis of extracts from various cell lines using Alix (E6P9B) Rabbit mAb.

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IP
Immunoprecipitation

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.

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IF-IC
Immunofluorescence (Immunocytochemistry)

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).

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Western Blotting Protocol

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.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 10X Tris Buffered Saline (TBS): (#12498) To prepare 1 L 1X TBS: add 100 ml 10X to 900 ml dH2O, mix.
  3. 1X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer. Dilute to 1X with dH2O.
  4. 10X Tris-Glycine SDS Running Buffer: (#4050) To prepare 1 L 1X running buffer: add 100 ml 10X running buffer to 900 ml dH2O, mix.
  5. 10X Tris-Glycine Transfer Buffer: (#12539) To prepare 1 L 1X Transfer Buffer: add 100 ml 10X Transfer Buffer to 200 ml methanol + 700 ml dH2O, mix.
  6. 10X Tris Buffered Saline with Tween® 20 (TBST): (#9997) To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH2O, mix.
  7. Nonfat Dry Milk: (#9999).
  8. Blocking Buffer: 1X TBST with 5% w/v nonfat dry milk; for 150 ml, add 7.5 g nonfat dry milk to 150 ml 1X TBST and mix well.
  9. Wash Buffer: (#9997) 1X TBST.
  10. Primary Antibody Dilution Buffer: 1X TBST with 5% nonfat dry milk; for 20 ml, add 1.0 g nonfat dry milk to 20 ml 1X TBST and mix well.
  11. Biotinylated Protein Ladder Detection Pack: (#7727).
  12. Prestained Protein Marker, Broad Range (11-190 kDa): (#13953).
  13. Blotting Membrane and Paper: (#12369) This protocol has been optimized for nitrocellulose membranes. Pore size 0.2 µm is generally recommended.
  14. Secondary Antibody Conjugated to HRP: Anti-rabbit IgG, HRP-linked Antibody (#7074).
  15. Detection Reagent: SignalFire™ ECL Reagent (#6883).

B. Protein Blotting

A general protocol for sample preparation.

  1. Treat cells by adding fresh media containing regulator for desired time.
  2. Aspirate media from cultures; wash cells with 1X PBS; aspirate.
  3. Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.
  4. Sonicate for 10–15 sec to complete cell lysis and shear DNA (to reduce sample viscosity).
  5. Heat a 20 µl sample to 95–100°C for 5 min; cool on ice.
  6. Microcentrifuge for 5 min.

  7. 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.

  8. Electrotransfer to nitrocellulose membrane (#12369).

C. Membrane Blocking and Antibody Incubations

NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.

I. Membrane Blocking

  1. (Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 min at room temperature.
  2. Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature.
  3. Wash three times for 5 min each with 15 ml of TBST.

II. Primary Antibody Incubation

  1. Incubate membrane and primary antibody (at the appropriate dilution and diluent as recommended in the product datasheet) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4°C.
  2. Wash three times for 5 min each with 15 ml of TBST.
  3. Incubate membrane with Anti-rabbit IgG, HRP-linked Antibody (#7074 at 1:2000) and Anti-biotin, HRP-linked Antibody (#7075 at 1:1000–1:3000) to detect biotinylated protein markers in 10 ml of blocking buffer with gentle agitation for 1 hr at room temperature.
  4. Wash three times for 5 min each with 15 ml of TBST.
  5. Proceed with detection (Section D).

D. Detection of Proteins

Directions for Use:

  1. Wash membrane-bound HRP (antibody conjugate) three times for 5 minutes in TBST.
  2. Prepare 1X SignalFire™ ECL Reagent (#6883)by diluting one part 2X Reagent A and one part 2X Reagent B (e.g. for 10 ml, add 5 ml Reagent A and 5 ml Reagent B). Mix well.
  3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.

* Avoid repeated exposure to skin.

posted June 2005

revised November 2013

Western Blot Reprobing Protocol

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.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.

  1. Wash Buffer: Tris Buffered Saline with Tween® 20 (TBST-10X) (#9997)
  2. Stripping Buffer: To prepare 100 ml, mix 6.25 ml of 1M Tris-HCl pH 6.8, 10 ml of 20% SDS and 700 μl β-mercaptoethanol. Bring to 100 ml with deionized H20. Make buffer fresh just prior to use.

B. Protocol

  1. After film exposure, wash membrane four times for 5 min each in TBST. Best results are obtained if the membrane is not allowed to dry.
  2. Incubate membrane for 30 min at 50°C in stripping buffer (with slight agitation).
  3. Wash membrane six times for 5 min each in TBST.
  4. (Optional) To assure that the original signal is removed, wash membrane twice for 5 min each with 10 ml of TBST. Incubate membrane with LumiGLO® with gentle agitation for 1 min at room temperature. Drain membrane of excess developing solution. Do not let dry. Wrap in plastic wrap and expose to x-ray film.
  5. Wash membrane again four times for 5 min each in TBST.
  6. The membrane is now ready to reuse. Start detection at the "Membrane Blocking and Antibody Incubations" step in the Western Immunoblotting Protocol.

posted June 2005

revised June 2016

Protocol Id: 263

PRINT

Immunoprecipitation for Native Proteins

This protocol is intended for immunoprecipitation of native proteins utilizing Protein A agarose beads for analysis by western immunoblot or kinase activity.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L of 1X PBS, add 50 ml 20X PBS to 950 ml dH2O, mix.

  2. 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.

  3. 3X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer.
  4. Protein A Agarose Beads: (#9863).
  5. 10X Kinase Buffer (for kinase assays): (#9802) To Prepare 1 ml of 1X kinase buffer, add 100 µl 10X kinase buffer to 900 µl dH2O, mix.
  6. ATP (10 mM) (for kinase assays): (#9804) To prepare 0.5 ml of ATP (200 µM), add 10 µl ATP (10 mM) to 490 µl 1X kinase buffer.

B. Preparing Cell Lysates

  1. Aspirate media. Treat cells by adding fresh media containing regulator for desired time.
  2. To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold 1X PBS.
  3. Remove PBS and add 0.5 ml ice-cold 1X cell lysis buffer to each plate (10 cm) and incubate on ice for 5 min.
  4. Scrape cells off the plate and transfer to microcentrifuge tubes. Keep on ice.
  5. Sonicate on ice three times for 5 sec each.
  6. Microcentrifuge for 10 min at 4°C, 14,000 x g and transfer the supernatant to a new tube. The supernatant is the cell lysate. If necessary, lysate can be stored at -80°C.

C. Immunoprecipitation

Cell Lysate Pre-Clearing (Optional)

  1. Vortex to mix beads.
  2. Add 10–30 µl of 50% Protein A agarose bead slurry to 200 µl cell lysate at 1 mg/ml.
  3. Incubate with rotation at 4°C for 30–60 min.
  4. Microcentrifuge for 10 min at 4°C. Transfer the supernatant to a fresh tube.
  5. Proceed to immunoprecipitation below.

Immunoprecipitation

  1. Add primary antibody (at the appropriate dilution as recommended in the product datasheet) to 200 µl cell lysate at 1 mg/ml. Incubate with rotation overnight at 4°C.
  2. Add protein A agarose (10–30 µl of 50% bead slurry). Incubate with rotation for 1–3 hr at 4°C.
  3. Microcentrifuge for 30 sec at 4°C. Wash pellet five times with 500 µl of 1X cell lysis buffer. Keep on ice between washes.
  4. Proceed to sample analysis by western immunoblotting or kinase activity (section D).

D. Sample Analysis

Proceed to one of the following specific set of steps.

For Analysis by Western Immunoblotting

  1. Resuspend the pellet with 20 µl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec at 14,000 x g.
  2. Heat the sample to 95–100°C for 2-5 min and microcentrifuge for 1 min at 14,000 x g.
  3. Load the sample (15–30 µl) on a 4–20% gel for SDS-PAGE.
  4. Analyze sample by western blot (see Western Immunoblotting Protocol).

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).

For Analysis by Kinase Assay

  1. Wash pellet twice with 500 µl 1X kinase buffer. Keep on ice.
  2. Suspend pellet in 40 µl 1X kinase buffer supplemented with 200 µM ATP and appropriate substrate.
  3. Incubate for 30 min at 30°C.
  4. Terminate reaction with 20 µl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec.
  5. Transfer supernatant containing phosphorylated substrate to another tube.
  6. Heat the sample to 95–100°C for 2–5 min and microcentrifuge for 1 min at 14,000 x g.
  7. Load the sample (15–30 µl) on SDS-PAGE (4–20%).

posted December 2008

revised April 2018

Protocol Id: 409

PRINT

Immunofluorescence (Immunocytochemistry)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.

  1. 20X Phosphate Buffered Saline (PBS): (9808) To prepare 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
  2. Formaldehyde: 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh and store opened vials at 4°C in dark, dilute in 1X PBS for use.
  3. Methanol, 100%
  4. Blocking Buffer (1X PBS / 5% normal serum / 0.3% Triton™ X-100): To prepare 10 ml, add 0.5 ml normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum (#5425)) and 0.5 mL 20X PBS to 9.0 mL dH2O, mix well. While stirring, add 30 µl Triton™ X-100.
  5. Antibody Dilution Buffer (1X PBS / 1% BSA / 0.3% Triton X-100): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1 g BSA (9998), mix.

  6. Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:

  7. Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).

B. Specimen Preparation - Cultured Cell Lines (IF-IC)

NOTE: Cells should be grown, treated, fixed and stained directly in multiwell plates, chamber slides or on coverslips.

  1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde in 1X PBS.
    NOTE: Formaldehyde is toxic, use only in fume hood.
  2. Allow cells to fix for 15 minutes at room temperature.
  3. Aspirate fixative, rinse three times in 1X PBS for 5 minutes each.
  4. Proceed with Immunostaining (Section C).

C. Immunostaining

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.

  1. Methanol Permeabilization Step: Cover cells with ice-cold 100% methanol (use enough to cover completely to a depth of 3–5 mm, DO NOT LET DRY), incubate in methanol for 10 minutes at –20°C, rinse in 1X PBS for 5 minutes.
  2. Block specimen in Blocking Buffer for 60 minutes.
  3. While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
  4. Aspirate blocking solution, apply diluted primary antibody.
  5. Incubate overnight at 4°C.
  6. Rinse three times in 1X PBS for 5 minutes each.
  7. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hours at room temperature in dark.
  8. Rinse in 1X PBS as in step 6.
  9. Coverslip slides with Prolong® Gold Antifade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).
  10. For best results examine specimens immediately using appropriate excitation wavelength. For long term storage, store slides flat at 4°C protected from light.

posted November 2006

revised December 2010

Protocol Id: 32

Application Dilutions
Western Blotting 1:1000
Immunoprecipitation 1:50
Immunofluorescence (Immunocytochemistry) 1:800
Storage:

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.

Species Reactivity:

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)

  1. Mattei, S. et al. (2006) Eur. J. Cell Biol. 85, 925-936.
  2. Odorizzi, G. (2006) J. Cell Sci. 119, 3025-3032.
  3. Vito, P. et al. (1999) J. Biol. Chem. 274, 1533-1540.
  4. Missotten, M. et al. (1999) Cell Death Differ. 6, 124-129.
  5. Katoh, K. et al. (2003) J. Biol. Chem. 278, 39104-39113.
  6. Sadoul, R. (2006) Biol. Cell 98, 69-77.
  7. Baietti, M.F. et al. (2012) Nat Cell Biol 14, 677-85.
  8. Chatellard-Causse, C. et al. (2002) J Biol Chem 277, 29108-15.
  9. Murrow, L. et al. (2015) Nat Cell Biol 17, 300-10.
Entrez-Gene Id
10015
Swiss-Prot Acc.
Q8WUM4
For Research Use Only. Not For Use In Diagnostic Procedures.

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XP is a registered trademark of Cell Signaling Technology, Inc.
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