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REACTIVITY SENSITIVITY MW (kDa) Isotype
H Endogenous Rabbit IgG
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Immunofluorescence (Immunocytochemistry)

Confocal immunofluorescent analysis of high-expressing 293T (left), HeLa (middle), or negative 786-0 (right) cells using PHGDH (D3D5E) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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Immunofluorescence (Immunocytochemistry)

A. Solutions and Reagents

Achieve higher quality immunofluorescent images using the efficient and cost-effective, pre-made reagents in our #12727 Immunofluorescence Application Solutions Kit

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade 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. 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.
  4. 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.
  5. Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:

  6. 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 4% formaldehyde diluted in 1X PBS.

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

  2. Allow cells to fix for 15 min at room temperature.
  3. Aspirate fixative, rinse three times in 1X PBS for 5 min 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 min.
  2. While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
  3. Aspirate blocking solution, apply diluted primary antibody.
  4. Incubate overnight at 4°C.
  5. Rinse three times in 1X PBS for 5 min each.
  6. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hr at room temperature in the dark.
  7. Rinse three times in 1X PBS for 5 min each.
  8. Coverslip slides with Prolong® Gold Antifade Reagent (#9071) or Prolong® Gold Antifade Reagent with DAPI (#8961).
  9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.

posted November 2006

revised November 2013

Protocol Id: 24

Product Usage Information

Application Dilutions
Immunofluorescence (Immunocytochemistry) 1:100

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.

Specificity / Sensitivity

PHGDH (D3D5E) Rabbit mAb recognizes endogenous levels of total PHGDH protein.


Species Reactivity: Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Val304 of human PHGDH protein.

Mammalian cells synthesize serine de novo by diverting a portion of the glycolytic intermediate 3-phosphoglycerate into the phosphorylated pathway of serine synthesis. This shift supports anabolism by providing precursors for the biosynthesis of proteins, nucleotides, creatine, porphyrins, phospholipids, and glutathione. Phosphoglycerate dehydrogenase (PHGDH) catalyzes the first step in the serine biosynthesis pathway by converting 3-phosphoglycerate into phosphohydroxy pyruvate (1).

Research studies demonstrate that an increase in serine biosynthesis supports growth and proliferation of cancer cells (2-4), which is supported by amplification and overexpression of PHGDH in a subset of melanoma and breast cancers (5,6). Suppression of PHGDH expression in cell lines with elevated PHGDH levels causes a strong decrease in cell proliferation and inhibits tumor growth in vivo (5). Additional evidence suggests that PHGDH interacts with and stabilizes FoxM1, which promotes the proliferation, invasion, and tumorigenicity of glioma cells (7).


1.  Locasale, J.W. (2013) Nat Rev Cancer 13, 572-83.

2.  Amelio, I. et al. (2013) Oncogene, [Epub ahead of print].

3.  Ma, L. et al. (2013) Cell 152, 599-611.

4.  Maddocks, O.D. et al. (2013) Nature 493, 542-6.

5.  Possemato, R. et al. (2011) Nature 476, 346-50.

6.  Locasale, J.W. et al. (2011) Nat Genet 43, 869-74.

7.  Liu, J. et al. (2013) J Neurooncol 111, 245-55.


Entrez-Gene Id 26227
Swiss-Prot Acc. O43175


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
DRAQ5 is a registered trademark of Biostatus Limited.

32770
PHGDH (D3D5E) Rabbit mAb (IF Formulated)