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REACTIVITY SENSITIVITY MW (kDa) SOURCE
H Endogenous 17 Rabbit

Western blot analysis of extracts from HeLa cells arrested in S phase or mitosis using Phospho-CENP-A (Ser7) Antibody (upper panel) or CENP-A Antibody #2186 (lower panel). S phase cells were treated for 12 hours with thymidine (2 mM), rinsed three times, released into normal growth medium for 10 hours and then treated an additional 12 hours with thymidine before harvesting. Mitotic cells were treated for 12 hours with thymidine, rinsed three times and then treated for 16 hours with paxitaxol (500 nM final).

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Confocal immunofluorescent analysis of a mitotic HeLa cell using Phospho-CENP-A (Ser7) Antibody (green fluorescence, appearing as white in the composite image) and β-Tubulin (9F3) Rabbit mAb (Alexa Fluor® 555 Conjugate) #2116 (red). Phospho-CENP-A signal is localized to bright spots in the metaphase plate. 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 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.

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. Bovine Serum Albumin (BSA): (#9998).
  11. Primary Antibody Dilution Buffer: 1X TBST with 5% BSA; for 20 ml, add 1.0 g BSA to 20 ml 1X TBST and mix well.
  12. Biotinylated Protein Ladder Detection Pack: (#7727).
  13. Prestained Protein Marker, Broad Range (Premixed Format): (#7720).
  14. Blotting Membrane and Paper: (#12369) This protocol has been optimized for nitrocellulose membranes. Pore size 0.2 µm is generally recommended.
  15. Secondary Antibody Conjugated to HRP: Anti-rabbit IgG, HRP-linked Antibody (#7074).
  16. 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 (#7720, 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

protocol id: 10

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 0.76 g Tris base, 2 g SDS and 700 μl β-mercaptoethanol. Bring to 100 ml with deionized H2O. Adjust pH to 6.8 with HCl.

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

Page

Immunoprecipitation for Native Proteins

This protocol is intended for immunoprecipitation of native proteins 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 Magnetic Beads: Use Protein A (#8687) for rabbit IgG immunoprecipitation.
  5. 6-Tube Magnetic Separation Rack: (#7017).
  6. 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.
  7. 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 magnetic bead slurry of to 200 µl cell lysate at 1 mg/ml.
  3. Incubate with rotation at 4°C for 30–60 min.
  4. Pellet beads using magnetic separation rack. 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 magnetic beads (10–30 µl of 50% bead slurry). Incubate with rotation for 10–30 min at 4°C.
  3. Pellet beads using magnetic separation rack. Wash pellet five times with 500 µl of 1X cell lysis buffer. Keep on ice between washes.
  4. Proceed to analyze 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 November 2013

protocol id: 410

Page

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 1 L 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, store opened vials at 4°C in dark, dilute in 1X PBS for use.
  3. Nonfat Dry Milk: (#9999).
  4. Phosphate Buffered Saline with 0.1% Triton™ X-100 (PBST): To prepare 1 L 1X PBST: add 50 ml 20X PBS and 1 mL Triton X-100 to 949 ml dH2O, mix well.
  5. Blocking Buffer (1X PBST with 5% w/v nonfat dry milk): To prepare 10 mL, add 0.5 g nonfat dry milk to 10 ml 1X PBST and mix well.
  6. Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:
    1. Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412
    2. Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 555 Conjugate) #4413
    3. Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 594 Conjugate) #8889
    4. Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4414
  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 multi-well plates, chamber slides or on coverslips.

  1. Aspirate liquid, then cover cells to a depth of 2-3 mm with 1% formaldehyde diluted in 1X PBS.

    NOTE: Formaldehyde is toxic, use only in fume hood.

  2. Allow cells to fix for 10 minutes at room temperature.
  3. Aspirate fixative, rinse three times in 1X PBST 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. Block specimen in Blocking Buffer for 60 minutes.
  2. While blocking, prepare primary antibody by diluting as indicated on datasheet in 1X PBST.
  3. Aspirate blocking solution, apply diluted primary antibody.
  4. Incubate overnight at 4°C.
  5. Rinse three times in 1X PBST for 5 minutes each.
  6. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in 1X PBST for 1-2 hours at room temperature in dark.
  7. Rinse three times in 1X PBST for 5 minutes each.
  8. Rinse briefly with 1X PBS, and coverslip slides with Prolong® Gold Antifade Reagent (#9071), or Prolong® Gold Antifade Reagent with DAPI (#8961).
  9. For best results, examine specimens immediately using appropriate excitation wavelength. For long-term storage, store slides flat at 4°C protected from light.

posted January 2009

revised December 2010

protocol id: 35

Product Usage Information

Application Dilutions
Western Blotting 1:1000
Immunoprecipitation 1:25
Immunofluorescence 1:100

Storage: 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.

Specificity / Sensitivity

Phospho-CENP-A (Ser7) Antibody detects endogenous levels of human CENP-A protein only when phosphorylated on Ser7. This antibody does not cross-react with other histone proteins, including Histone H3.


Species Reactivity: Human
Species predicted to react based on 100% sequence homology: Monkey

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser7 of human CENP-A protein. Antibodies are purified by peptide affinity chromatography.

Modulation of chromatin structure plays a critical role in the regulation of transcription and replication of the eukaryotic genome. The nucleosome, made up of four core histone proteins (H2A, H2B, H3, and H4), is the primary building block of chromatin. In addition to the growing number of post-translational histone modifications regulating chromatin structure, cells can also exchange canonical histones with variant histones that can directly or indirectly modulate chromatin structure (1). CENP-A, also known as the chromatin-associated protein CSE4 (capping-enzyme suppressor 4-p), is an essential histone H3 variant that replaces canonical histone H3 in centromeric heterochromatin (2). The greatest divergence between CENP-A and canonical histone H3 occurs in the amino-terminal tail of the protein, which binds linker DNA between nucleosomes and facilitates proper folding of centromeric heterochromatin (3). The amino-terminal tail of CENP-A is also required for recruitment of other centromeric proteins (CENP-C, hSMC1, hZW10), proper kinetochore assembly and chromosome segregation during mitosis (4). Additional sequence divergence in the histone fold domain is responsible for correct targeting of CENP-A to the centromere (5). Many of the functions of CENP-A are regulated by phosphorylation (6,7). Aurora A-dependent phosphorylation of CENP-A on Ser7 during prophase is required for proper targeting of Aurora B to the inner centromere in prometaphase, proper kinetochore/microtubule attachment and proper alignment of chromosomes during mitosis (6).


1.  Jin, J. et al. (2005) Trends Biochem Sci 30, 680-7.

2.  Ausió, J. (2006) Brief Funct Genomic Proteomic 5, 228-43.

3.  Heit, R. et al. (2006) Biochem Cell Biol 84, 605-18.

4.  Van Hooser, A.A. et al. (2001) J Cell Sci 114, 3529-42.

5.  Black, B.E. et al. (2004) Nature 430, 578-82.

6.  Kunitoku, N. et al. (2003) Dev Cell 5, 853-64.

7.  Zeitlin, S.G. et al. (2001) J Cell Biol 155, 1147-57.


Entrez-Gene Id 1058
Swiss-Prot Acc. P49450

Product Specific References

Sun, L. et al. (2010) J Pathol 221, 425-32.


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.
Alexa Fluor® is a registered trademark of Life Technologies Corporation.
DRAQ5® is a registered trademark of Biostatus Limited.