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- Table of Contents
STAT5A / Signal transducer and activator of transcription 5A · Western blot design guide
Design a Western Blot for STAT5A
Real validated STAT5A Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-STAT5A 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
STAT5A 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 | ~90.6 kDa | |
| Observed band | ~95 kDa | |
| Gel | 5–20% (catalog PA1840) | |
| Positive control | Lymph node (IHC candidate; verify WB) +4 more | |
| Negative control | Bronchus (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Ubl conjugation | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated STAT5A Western Blot Protocols
The PA1840 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Hela, human K562, human Raji (catalog PA1840) |
| Gel % | 5–20% (catalog PA1840) |
| Load | 30 ug; reducing conditions (catalog PA1840) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog PA1840) |
| Membrane | nitrocellulose membrane (catalog PA1840) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog PA1840) |
| Primary antibody | PA1840 · 0.5 μg/mL (catalog PA1840) |
| Primary incubation | overnight at 4°C (catalog PA1840) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog PA1840) |
| Secondary incubation | 1.5 hour at RT (catalog PA1840) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog PA1840) |
| Detection | ECL (catalog PA1840) |
Section 2
What Is the Expected STAT5A Western Blot Band Size?
STAT5A is predicted at 90.6 kDa and observed at ~95 kDa; the cause of the difference is not established.
| Band at ~95 kDa | Matches the empirical STAT5A band; confirm identity with ordinary antibody controls. |
| Band near 90.6 kDa | Near the predicted mass of full-length STAT5A; migration can differ from calculated mass. |
| Multiple nearby bands | Could reflect isoforms 1 and 2 or phosphorylation states; distinct migration is unproven. |
| Band near twice the monomer size | Could reflect a STAT5A homodimer if sample conditions preserve the complex. |
💡Expected STAT5A appearanceSTAT5A has a predicted mass of 90.6 kDa and an empirical band at ~95 kDa; the cause of the difference is unestablished, so confirm band identity with appropriate controls.
| Predicted STAT5A mass | 90.6 kDa is the calculated mass; the empirical band is ~95 kDa. |
| Isoforms 1 and 2 | Alternative splicing could change size, but their relative masses and migration are unspecified. |
| STAT5A homodimer | Could appear near twice the monomer size if the complex survives sample preparation. |
| Phosphotyrosine at residues 90, 682, and 694 | Phosphorylation could affect migration, but a visible shift is not established. |
| Phosphoserine at residues 128, 193, and 780 | Phosphorylation could affect migration, but a visible shift is not established. |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | A retained STAT5A homodimer is possible; the ~95 kDa band is also above calculated mass for an unestablished reason. | Compare denaturing conditions and confirm band identity. |
| Band lower than expected | An alternative splice product is possible, but isoform masses are unspecified. | Verify with an independent STAT5A antibody or isoform-specific evidence. |
| Multiple bands | Isoforms or phosphorylation states are possible, but distinct migration is unproven. | Compare antibody controls and, if testing phosphorylation, phosphatase-treated samples. |
| Weak or no signal | STAT5A may be below the detection limit in the tested sample. | Check sample loading and antibody performance with a positive lysate. |
| Fragments below expected size | STAT5A may have degraded during sample handling. | Prepare fresh lysate with protease inhibitors and confirm fragment identity. |
Experiment evidence
Western blot images showing STAT5A band
PA1840 · STAT5A
P01087-1 · STAT5A
M01087-1 · STAT5A
M01087 · STAT5A
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for STAT5A Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for STAT5A in Western blot, you can use lymph node tissue, which shows high expression in HPA.
Positive control: Lymph node (IHC candidate; verify WB)
Negative control: Bronchus (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: Whole-cell lysates should capture STAT5A, though phosphorylation can shift it from cytoplasm to nucleus.
HPA tissue expression evidence for STAT5A
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 |
|---|---|---|---|---|
| Lymph node | germinal center cells | High | Protein (IHC) | HPA → |
| Tonsil | non-germinal center cells | High | Protein (IHC) | HPA → |
| Adipose tissue | adipocytes | Medium | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | lymphoid tissue | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Bronchus | respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Caudate | glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Cerebral cortex | endothelial cells | Not detected | Protein (IHC) | HPA → |
| Cervix | glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced STAT5A Western Blot Tips
Deeper troubleshooting and optimisation questions for STAT5A, answered from its protein features.
How should STAT5A 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 STAT5A isoforms produce different bands?
Isoforms · UniProt lists two isoforms. Isoform 2 lacks residues 96–125 of the canonical sequence, so it could be smaller. Whether the isoforms resolve as separate bands depends on the antibody and gel; the features do not establish their abundance.
Which STAT5A phosphorylation sites matter when interpreting bands?
PTM · UniProt annotates phosphotyrosines at 90, 682 and 694, with Tyr694 phosphorylated by JAK2, and phosphoserines at 128, 193 and 780. These are UniProt canonical-sequence coordinates; antibody or paper numbering may differ. Phosphorylation alone does not guarantee a visible mobility shift.
Measure phospho-STAT5A and total STAT5A in matched samples, then compare their ratio across conditions. Nuclear translocation in response to phosphorylation also makes consistent sample fractionation important. Report which phosphosite the antibody detects, using UniProt coordinates when referring to the listed sites.
Measure phospho-STAT5A and total STAT5A in matched samples, then compare their ratio across conditions. Nuclear translocation in response to phosphorylation also makes consistent sample fractionation important. Report which phosphosite the antibody detects, using UniProt coordinates when referring to the listed sites.
Does this guide establish induction of STAT5A?
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 STAT5A?
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 PA1840 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 STAT5A be quantified?
Quantitation · Standard workflow guidance: quantify only a validated target band within the linear exposure range. Use consistent sample preparation and loading, retain biological replicates, and avoid interpreting saturation or loading differences as regulation.
Why does STAT5A run near 95 kDa instead of 90.6 kDa?
Interpretation · The predicted mass is 90.6 kDa, while the reported apparent band is about 95 kDa. STAT5A has annotated phosphorylation sites, but their presence alone does not establish why it migrates at 95 kDa or demonstrate a visible shift.
UniProt identifies JAK2 phosphorylation at Tyr694 and says STAT5A translocates to the nucleus in response to phosphorylation. Collect matched samples at defined times after the experimental stimulus and compare phospho-STAT5A with total STAT5A. The supplied features do not specify a stimulus or response time.
Check whether the antibody recognizes both isoforms: isoform 2 lacks canonical residues 96–125. Compare total and site-specific phospho blots for condition-dependent signals. The listed phosphorylation sites do not, by themselves, prove a band shift, and the supplied features provide no basis for assigning another band to cleavage or glycosylation.
UniProt identifies JAK2 phosphorylation at Tyr694 and says STAT5A translocates to the nucleus in response to phosphorylation. Collect matched samples at defined times after the experimental stimulus and compare phospho-STAT5A with total STAT5A. The supplied features do not specify a stimulus or response time.
Check whether the antibody recognizes both isoforms: isoform 2 lacks canonical residues 96–125. Compare total and site-specific phospho blots for condition-dependent signals. The listed phosphorylation sites do not, by themselves, prove a band shift, and the supplied features provide no basis for assigning another band to cleavage or glycosylation.
Boster reagents
STAT5A 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
Anti-STAT5a Antibody Picoband®
Cat # PA1840
Real WB data
Anti-Phospho-Stat5 (Y694) STAT5A Rabbit Monoclonal Antibody
Cat # P01087-1
Real WB data
Anti-STAT5a Rabbit Monoclonal Antibody
Cat # M01087-1
Real WB data
Anti-STAT5A/B Rabbit Monoclonal Antibody
Cat # M01087
All four listed antibodies have Western blot images. PA1840, M01087-1, and M01087 show bands in human, mouse, and rat samples; P01087-1 shows a band in human K562 cells. These examples do not establish performance in every sample or phospho-specific validation.
Which to pick: For STAT5A, PA1840 and M01087-1 have images with human, mouse, and rat samples. Choose based on the sample closest to yours. P01087-1 is labeled phospho-STAT5 (Y694), but its supplied image alone does not verify phospho specificity; M01087 is labeled STAT5A/B.
Source: BosterBio STAT5A gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P42229.
- Human Protein Atlas. STAT5A tissue expression.
- PMC6270101 — target-verified WB comparison
- PMC6270553 — target-verified WB comparison
- PMC11271408 — target-verified WB comparison