PHGDH / D-3-phosphoglycerate dehydrogenase · Western blot design guide

Design a Western Blot for PHGDH

Real validated PHGDH Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-PHGDH WB antibodies. Everything you need to plan the experiment before you commit precious samples.

Evidence assembled September 2026 · For research use; verify linked source records and product datasheet before use

Western blot protocol sheet for PHGDH: expected band ~56.7 kDa, hero antibody A03355-1, catalog values and labelled standard workflow; separate PMC comparisons on the guide — Printable **PHGDH** Western blot protocol sheet — expected band ~56.7 kDa, antibody A03355-1, controls and PMC citations. Open the full PHGDH WB guide →

Navigate to: Protocols Band Controls Antibodies

PHGDH Western Blot Experimental Design Guide

Expected bands, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.

Must know before running

	Expected band	~56.7 kDa
	Observed band	~57 kDa
	Gel	5–20% (catalog A03355-1)
	Positive control ⓘ	Appendix (IHC candidate; verify WB) +4 more
	Negative control ⓘ	Suggested KO / knockdown lysate

Important caveats

Reasons your observed band may differ from the expected size.

	PTM	Phosphorylated + Acetylated
	Caveat	Modification-state controls
	Gene-set association	MSigDB Hallmark membership
	Isoform	1 isoform(s)

Section 1

Real Curated PHGDH Western Blot Protocols

The A03355-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.

Recommended Western blot protocol parameters
Sample / lysate	human U251, human Jurkat, human HEL (catalog A03355-1)
Gel %	5–20% (catalog A03355-1)
Load	30 ug; reducing conditions (catalog A03355-1)
Transfer	a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A03355-1)
Membrane	nitrocellulose membrane (catalog A03355-1)
Blocking	5% non-fat milk/TBS for 1.5 hour at RT (catalog A03355-1)
Primary antibody	A03355-1 · 0.5 μg/mL (catalog A03355-1)
Primary incubation	overnight at 4°C (catalog A03355-1)
Secondary antibody	goat anti-rabbit IgG-HRP, 1:5000 (catalog A03355-1)
Secondary incubation	1.5 hour at RT (catalog A03355-1)
Wash	TBS-0.1%Tween 3 times with 5 minutes each (catalog A03355-1)
Detection	ECL (catalog A03355-1)

Section 2

What Is the Expected PHGDH Western Blot Band Size?

PHGDH is predicted at 56.7 kDa and observed at ~57 kDa; no distinct cause for the small difference is established.

What am I looking at on my blot?
Band at ~57 kDa	Matches the reported PHGDH band and its 56.7 kDa predicted mass
Single sharp band near ~57 kDa	Consistent with the reported PHGDH band; listed modifications need not produce visible shifts
Close doublet near ~57 kDa	PHGDH has phosphorylation and acetylation sites, but their contribution to a doublet is unproven
Additional band above ~57 kDa	PHGDH identity requires confirmation; its listed modifications do not establish this shift

💡Expected PHGDH appearancePHGDH has a predicted mass of 56.7 kDa and an empirical band at ~57 kDa; confirm band identity with an appropriate positive control and antibody specificity controls.

How each factor affects band size
Predicted mass of 56.7 kDa	Provides the reference size for the ~57 kDa empirical band
N-acetylalanine at residue 2	Adds a small modification; a visible migration shift is not established
Phosphoserine at residue 14	Adds a phosphate; a visible migration shift is not established
N6-acetyllysine at residue 58	Adds a small modification; a visible migration shift is not established
Phosphothreonine at residue 78	Adds a phosphate; a visible migration shift is not established

Why is my band missing or off?
Situation	Likely cause	Next action
No band in lysate	PHGDH may be below detection in the sample	Compare with a Jurkat or U251 whole-cell lysate positive control
Band higher than expected	The listed modifications do not establish a large upward shift	Check antibody specificity and compare with the ~57 kDa positive-control band
Band lower than expected	No signal peptide or propeptide cleavage is listed	Check band identity against a positive control and assess sample degradation
Multiple bands	Modification sites are listed, but distinct migrating forms are unproven	Identify the ~57 kDa band with a positive control and test additional bands for specificity
Weak or no signal	PHGDH may be below detection or antibody detection may be insufficient	Check the positive control and review sample loading and antibody detection
Fragments below expected size	Sample degradation is possible; no defined cleavage fragments are supplied	Review sample handling and compare with a fresh positive-control lysate

Source: UniProt O43175 Human Protein Atlas product datasheet (A03355-1)

Experiment evidence

Western blot images showing PHGDH band

A03355-1 · PHGDH

← swipe / scroll for more images →

Real WB data behind the band-size expectations in this section.

Sample controls for PHGDH Western blot

🧪HPA-IHC candidate guidance (verify in WB): For positive controls for PHGDH in Western blot, you can use appendix tissue, which HPA scores High for PHGDH.

Positive control: Appendix (IHC candidate; verify WB)

Negative control: Suggested KO / knockdown lysate

Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.

⚠️Feasibility: HPA lists no Not-detected tissue, so use siRNA knockdown or a KO line for a clean negative control.

HPA tissue expression evidence for PHGDH

Comprehensive Human Protein Atlas IHC scoring per tissue. Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.

Higher expression tissues · candidate positive controls from IHC

Tissue	Cell type	Level	Evidence	Source
Appendix	glandular cells	High	Protein (IHC)	HPA →
Breast	glandular cells	High	Protein (IHC)	HPA →
Bronchus	basal cells	High	Protein (IHC)	HPA →
Caudate	glial cells	High	Protein (IHC)	HPA →
Cerebral cortex	endothelial cells	High	Protein (IHC)	HPA →

Lower expression tissues · IHC evidence, not confirmed WB-negative controls

Tissue	Cell type	Level	Evidence	Source
Adrenal gland	glandular cells	Low	Protein (IHC)	HPA →
Bone marrow	hematopoietic cells	Low	Protein (IHC)	HPA →
Heart muscle	cardiomyocytes	Low	Protein (IHC)	HPA →
Lung	alveolar cells	Low	Protein (IHC)	HPA →
Placenta	trophoblastic cells	Low	Protein (IHC)	HPA →

Section 3

Advanced PHGDH Western Blot Tips

Deeper troubleshooting and optimisation questions for PHGDH, answered from its protein features.

How should PHGDH band migration be interpreted?

Band shift · Use the separately labelled calculated mass and catalog-observed evidence above. A sequence annotation does not establish an observed migration shift. Verify target identity with orthogonal controls.

Could PHGDH isoforms explain multiple bands?

Isoforms · Only one isoform is listed, with no alternative sequence. These features do not support assigning additional bands to known PHGDH isoforms.

Which PHGDH phosphorylation sites matter when interpreting bands?

PTM · UniProt lists phosphoserine at position 14 and phosphothreonine at position 78. These are UniProt sequence coordinates; paper or antibody numbering may differ. Their presence does not establish a visible band shift.

UniProt lists N-acetylalanine at position 2, alternate N6-acetyllysine at position 21, and N6-acetyllysine at position 58. Use these UniProt coordinates when checking site-specific reagents; do not silently substitute another numbering convention.

Does this guide establish induction of PHGDH?

Induction · No general induction response is established by this guide. A pathway or gene-set association is not evidence of induction in a particular specimen. Verify the relevant treatment and control in a target-specific experiment.

How should transfer be checked for PHGDH?

Transfer · Standard workflow guidance: verify transfer efficiency for the intended target size before interpreting a weak signal. Use total-protein assessment and optimize transfer for the membrane, gel and apparatus; the labelled catalog values take precedence.

How should blocking be optimized?

Blocking · Standard workflow guidance: follow the A03355-1 datasheet where specified. Otherwise compare 5% milk or 5% BSA in TBST; for a phospho-specific assay start with BSA. Optimize background and specific signal with matched controls.

How should PHGDH abundance be quantified?

Quantitation · Quantify the band near the expected 56.7 kDa mass consistently across samples. PHGDH is an enzyme in serine biosynthesis with listed phosphorylation and acetylation sites, so total band intensity alone does not establish enzyme activity or modification state.

Why is the PHGDH band near 57 kDa?

Interpretation · The observed band near 57 kDa closely matches the predicted 56.7 kDa mass. The listed modifications alone do not establish a visible shift or explain a mass difference.

Check the expected ~57 kDa band first. PHGDH is annotated as a homotetramer, but that annotation alone does not identify a higher Western-blot band as a tetramer. No signal peptide, propeptide, glycosylation site, or alternative sequence is listed to explain additional bands.

Boster reagents

PHGDH Western Blot Antibodies

Catalog antibodies with Western blot application and product-specific WB images. Evaluate suitability with the reported sample, controls and experimental conditions.

Real WB data

Western blot analysis of PHGDH using anti-PHGDH antibody (A03355-1). Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions. Lane 1: human U251 whole cell lysates, Lane 2: human Jurkat whole cell lysates, Lane 3: human HEL whole cell lysates, Lane 4: human K562 whole cell lysates, Lane 5: rat liver tissue lysates, Lane 6: rat kidney tissue lysates, Lane 7: mouse liver tissue lysates, Lane 8: mouse kidney tissue lysates. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-PHGDH antigen affinity purified polyclonal antibody (A03355-1) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for PHGDH at approximately 57 kDa. The expected band size for PHGDH is at 57 kDa.

Anti-PHGDH Antibody Picoband®

Cat # A03355-1

View product →

The catalog reports A03355-1 for PHGDH Western blotting. Its product image reports a band near the expected 57 kDa in human cell lines and rat and mouse liver and kidney lysates. This is product evidence; no independent validation is supplied.

Which to pick: A03355-1 is the only listed antibody. It reports human, mouse, and rat reactivity and includes a Western blot image for those sample contexts. Use its reported 57 kDa band and assay conditions as a starting point.

Source: BosterBio PHGDH gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.

References

UniProt Consortium. UniProt entry O43175.
Human Protein Atlas. PHGDH tissue expression.
PMC12885894 — target-verified WB comparison
PMC12243184 — target-verified WB comparison
PMC4414297 — target-verified WB comparison