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      H M R Mk
REACTIVITY SENSITIVITY MW (kDa)
Endogenous 82 to 97

Western blot analysis of extracts from U-937 cells using BAFF (D7I1U) Rabbit mAb.

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Western blot analysis of 1 ng recombinant Human BAFF/TNFSF13B (hBAFF) #5233 using BAFF (D7I1U) Rabbit mAb.

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Immunohistochemical analysis of paraffin-embedded human lymphoma using CD79A (D1X5C) XP® Rabbit mAb.

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Immunohistochemical analysis of paraffin-embedded human breast carcinoma using CD79A (D1X5C) XP® Rabbit mAb.

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Immunohistochemical analysis of paraffin-embedded human lung carcinoma using CD79A (D1X5C) XP® Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).

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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

Immunohistochemistry (Paraffin)

A. Solutions and Reagents

  1. Xylene
  2. Ethanol, anhydrous denatured, histological grade (100% and 95%)
  3. Deionized water (dH2O)
  4. Hematoxylin (optional)
  5. Wash Buffer:
    1X TBS/0.1% Tween-20 (1X TBST): To prepare 1 L add 100 ml 10X TBS to 900 ml dH2O. Add 1 ml Tween-20 and mix.
    10X Tris Buffered Saline (TBS): To prepare 1 L add 24.2 g Trizma® base (C4H11NO3) and 80 g sodium chloride (NaCl) to 1 L dH2O. Adjust pH to 7.6 with concentrated HCl.
  6. *Antibody Diluent:
    1. SignalStain® Antibody Diluent #8112
    2. TBST/5% normal goat serum (#5425): To 5 ml 1X TBST add 250 µl normal goat serum.
    3. PBST/5% normal goat serum (#5425): To 5 ml 1X PBST add 250 µl normal goat serum.
      1X PBS/0.1% Tween-20 (1X PBST): To prepare 1 L add 100 ml 10X PBS to 900 ml dH2O. Add 1 ml Tween-20 and mix.
      10X Phosphate Buffered Saline (PBS): To prepare 1 L add 80 g sodium chloride (NaCl), 2 g potassium chloride (KCl), 14.4 g sodium phophate, dibasic (Na2HPO4) and 2.4 g potassium phosphate, monobasic (KH2PO4) to 1 L dH2O. Adjust pH to 7.4.
  7. *Antigen Unmasking:
    1. Citrate: 10 mM Sodium Citrate Buffer: To prepare 1 L add 2.94 g sodium citrate trisodium salt dihydrate (C6H5Na3O7•2H2O) to 1 L dH2O. Adjust pH to 6.0.
    2. EDTA: 1 mM EDTA: To prepare 1 L add 0.372 g EDTA (C10H14N2O8Na2•2H2O) to 1 L dH2O. Adjust pH to 8.0.
    3. TE: 10 mM Tris/1 mM EDTA, pH 9.0: To prepare 1L add 1.21 g Trizma® base (C4H11NO3) and 0.372 g EDTA (C10H14N2O8Na2•2H2O) to 950 ml dH2O. Adjust pH to 9.0, then adjust final volume to 1000 ml with dH2O.
    4. Pepsin: 1 mg/ml in Tris-HCl pH 2.0.
  8. 3% Hydrogen Peroxide: To prepare, add 10 ml 30% H2O2 to 90 ml dH2O.
  9. Blocking Solution: TBST/5% normal goat serum (#5425): to 5 ml 1X TBST add 250 µl normal goat serum.
  10. Biotinylated secondary antibody.
  11. ABC Reagent: (Vectastain ABC Kit, Vector Laboratories, Inc., Burlingame, CA) Prepare according to manufacturer’s instructions 30 minutes before use.
  12. DAB Reagent or suitable substrate: Prepare according to manufacturer’s recommendations.

B. Deparaffinization/Rehydration

NOTE: Do not allow slides to dry at any time during this procedure.

  1. Deparaffinize/hydrate sections:
    1. Incubate sections in three washes of xylene for 5 minutes each.
    2. Incubate sections in two washes of 100% ethanol for 10 minutes each.
    3. Incubate sections in two washes of 95% ethanol for 10 minutes each.
  2. Wash sections twice in dH2O for 5 minutes each.

C. *Antigen Unmasking

NOTE: Consult product data sheet for specific recommendation for the unmasking solution.

  1. For Citrate: Bring slides to a boil in 10 mM sodium citrate buffer pH 6.0 then maintain at a sub-boiling temperature for 10 minutes. Cool slides on bench top for 30 minutes.
  2. For EDTA: Bring slides to a boil in 1 mM EDTA pH 8.0 followed by 15 minutes at a sub-boiling temperature. No cooling is necessary.
  3. For TE: Bring slides to a boil in 10 mM TE/1 mM EDTA, pH 9.0 then maintain at a sub-boiling temperature for 18 minutes. Cool on the bench for 30 minutes.
  4. For Pepsin: Digest for 10 minutes at 37°C.

D. Staining

  1. Wash sections in dH2O three times for 5 minutes each.
  2. Incubate sections in 3% hydrogen peroxide for 10 minutes.
  3. Wash sections in dH2O twice for 5 minutes each.

NOTE: Consult product data sheet for recommended antibody diluent.

  1. Wash sections in wash buffer for 5 minutes.
  2. Block each section with 100-400 µl blocking solution for 1 hour at room temperature.
  3. Remove blocking solution and add 100-400 µl primary antibody diluted in recommended antibody diluent to each section. Incubate overnight at 4°C.
  4. Remove antibody solution and wash sections in wash buffer three times for 5 minutes each.
  5. Add 100-400 µl biotinylated secondary antibody, diluted in TBST per manufacturer’s recommendation, to each section. Incubate 30 minutes at room temperature.
  6. If using ABC avidin/biotin method, prepare ABC reagent according to the manufacturer’s instructions and incubate solution for 30 minutes at room temperature.
  7. Remove secondary antibody solution and wash sections three times with wash buffer for 5 minutes each.
  8. Add 100-400 µl ABC reagent to each section and incubate for 30 minutes at room temperature.
  9. Remove ABC reagent and wash sections three times in wash buffer for 5 minutes each.
  10. Add 100-400 µl DAB or suitable substrate to each section and monitor staining closely.
  11. As soon as the sections develop, immerse slides in dH2O.
  12. If desired, counterstain sections in hematoxylin per manufacturer’s instructions.
  13. Wash sections in dH2O two times for 5 minutes each.
  14. Dehydrate sections:
    1. Incubate sections in 95% ethanol two times for 10 seconds each.
    2. Repeat in 100% ethanol, incubating sections two times for 10 seconds each.
    3. Repeat in xylene, incubating sections two times for 10 seconds each.
  15. Mount coverslips.

posted June 2005

revised February 2008

protocol id: 26

Product Usage Information

Application Dilutions
Western Blotting 1:1000
Immunohistochemistry 1:250

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

The Fox03a Antibody detects exogenous and endogenous levels of total Fox03a protein. The antibody does not detect the exogenously expressed family members Fox04 or Fox01. The antibody reacts with denatured components of bovine serum including BSA.


Species predicted to react based on 100% sequence homology: Mouse, Rat

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the sequence of human Fox03a. Antibodies are purified by protein A and peptide affinity chromatography.

The Forkhead family of transcription factors is involved in tumorigenesis of rhabdomyosarcoma and acute leukemias (1-3). Within the family, three members (FoxO1, FoxO4, and FoxO3a) have sequence similarity to the nematode orthologue DAF-16, which mediates signaling via a pathway involving IGFR1, PI3K, and Akt (4-6). Active forkhead members act as tumor suppressors by promoting cell cycle arrest and apoptosis. Increased expression of any FoxO member results in the activation of the cell cycle inhibitor p27 Kip1. Forkhead transcription factors also play a part in TGF-β-mediated upregulation of p21 Cip1, a process negatively regulated through PI3K (7). Increased proliferation results when forkhead transcription factors are inactivated through phosphorylation by Akt at Thr24, Ser256, and Ser319, which results in nuclear export and inhibition of transcription factor activity (8). Forkhead transcription factors can also be inhibited by the deacetylase sirtuin (SirT1) (9).


In Fox03a, the three sites phosphorylated by Akt mentioned above are Thr32, Ser253 and Ser315. Fox03a associates with 14-3-3 proteins upon phosphorylation by Akt and is retained in the cytoplasm. In the absence of survival factors, Fox03a is dephosphorylated, translocates to the nucleus and triggers cell death by a Fas ligand-dependent mechanism (7).


1.  Anderson, M.J. et al. (1998) Genomics 47, 187-99.

2.  Galili, N. et al. (1993) Nat Genet 5, 230-5.

3.  Borkhardt, A. et al. (1997) Oncogene 14, 195-202.

4.  Nakae, J. et al. (1999) J Biol Chem 274, 15982-5.

5.  Rena, G. et al. (1999) J Biol Chem 274, 17179-83.

6.  Guo, S. et al. (1999) J Biol Chem 274, 17184-92.

7.  Seoane, J. et al. (2004) Cell 117, 211-23.

8.  Arden, K.C. (2004) Mol Cell 14, 416-8.

9.  Yang, Y. et al. (2005) EMBO J 24, 1021-32.


Entrez-Gene Id 2309
Swiss-Prot Acc. O43524


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