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- Table of Contents
PYGM / Glycogen phosphorylase, muscle form · Western blot design guide
Design a Western Blot for PYGM
Source-linked PYGM Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-PYGM 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
PYGM Western Blot Experimental Design Guide
Expected bands, source-linked protocol options, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before running
| Expected band | ~97.1 kDa | |
| Observed band | ~97 kDa | |
| Gel | 8% (catalog A02971-1) | |
| Positive control | Skeletal muscle (IHC candidate; verify WB) | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Acetylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Source-Linked PYGM Western Blot Protocol Options
The A02971-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | rat heart, mouse heart (catalog A02971-1) |
| Gel % | 8% (catalog A02971-1) |
| Load | 30 ug; reducing conditions (catalog A02971-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A02971-1) |
| Membrane | nitrocellulose membrane (catalog A02971-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A02971-1) |
| Primary antibody | A02971-1 · 0.5 μg/mL (catalog A02971-1) |
| Primary incubation | overnight at 4°C (catalog A02971-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A02971-1) |
| Secondary incubation | 1.5 hour at RT (catalog A02971-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A02971-1) |
| Detection | ECL (catalog A02971-1) |
Section 2
What Is the Expected PYGM Western Blot Band Size?
PYGM is predicted at 97.1 kDa and observed near 97 kDa; its annotated isoforms and homodimer do not establish additional bands.
| Band near 97 kDa | Matches the reported PYGM band and predicted monomer size |
| Band near 194 kDa | Could reflect a homodimer that persists during sample preparation; confirm its identity |
| Several bands near 97 kDa | Could reflect isoforms 1 and 2, though their migration difference is unestablished |
| Close doublet near 97 kDa | Could reflect phosphorylation states; confirm with a phosphorylation-sensitive control |
💡Expected PYGM appearancePYGM has a predicted monomer mass of 97.1 kDa and an empirical band near 97 kDa; confirm band identity with an appropriate antibody control.
| Predicted PYGM monomer mass | 97.1 kDa predicted; the reported band is near 97 kDa |
| Homodimer formation | Could yield a band near twice the monomer size if the complex survives sample preparation |
| Isoform 1 | May migrate differently from isoform 2; its individual mass is not supplied |
| Isoform 2 | May migrate differently from isoform 1; its individual mass is not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | A PYGM homodimer may persist during sample preparation | Compare denaturing conditions and verify the band with an independent PYGM antibody |
| Band lower than expected | The band may be a fragment or an unrelated antibody target | Use an independent PYGM antibody and check sample integrity |
| Multiple bands | Isoforms 1 and 2 are annotated, but distinct migration is unestablished | Check each band's identity with an independent antibody or isoform-specific control |
| Weak or no signal | Too little detectable PYGM or inefficient transfer of the approximately 97 kDa protein | Check sample loading, transfer, and a positive control |
| Fragments below expected size | Protein degradation during sample preparation is possible | Prepare fresh samples with protease inhibitors and compare with a positive control |
Experiment evidence
Western blot images showing PYGM band
A02971-1 · PYGM
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for PYGM Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for PYGM in Western blot, you can use skeletal muscle lysate.
Positive control: Skeletal muscle (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: HPA reports high expression in skeletal muscle and no detection in adipose tissue, making tissue controls feasible.
HPA tissue expression evidence for PYGM
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 |
|---|---|---|---|---|
| Skeletal muscle | myocytes | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | endocrine cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced PYGM Western Blot Tips
Deeper troubleshooting and optimisation questions for PYGM, answered from its protein features.
How should PYGM 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 PYGM isoforms produce different bands?
Isoforms · UniProt lists two isoforms. Isoform 2 lacks residues 82–169 of the canonical sequence, so a lower molecular weight band is plausible if it is expressed and recognized by the antibody. The supplied features do not establish that such a band appears on a blot.
Which PYGM phosphorylation site marks phosphorylase A?
PTM · UniProt assigns PHK-mediated phosphorylation to Ser15 in phosphorylase A. Ser15 uses the supplied UniProt numbering; paper or antibody numbering may differ. A phospho-specific antibody directed at this site can assess that modification, while a total PYGM antibody measures PYGM regardless of Ser15 phosphorylation.
UniProt lists N-acetylserine at position 2, phosphosites at 15, 204, 227, 430, 473, 514, 747 and 748, and pyridoxal phosphate attachment at Lys681. These features alone do not demonstrate a resolvable Western blot shift. Compare matched samples directly before assigning a shifted band to a modification.
Measure the phospho-specific signal and total PYGM in the same samples, then compare their ratio across conditions. This distinguishes a change in phosphorylation from a change in PYGM abundance. For phosphorylase A, target the PHK-dependent Ser15 site using the supplied UniProt numbering.
UniProt lists N-acetylserine at position 2, phosphosites at 15, 204, 227, 430, 473, 514, 747 and 748, and pyridoxal phosphate attachment at Lys681. These features alone do not demonstrate a resolvable Western blot shift. Compare matched samples directly before assigning a shifted band to a modification.
Measure the phospho-specific signal and total PYGM in the same samples, then compare their ratio across conditions. This distinguishes a change in phosphorylation from a change in PYGM abundance. For phosphorylase A, target the PHK-dependent Ser15 site using the supplied UniProt numbering.
Does this guide establish induction of PYGM?
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 PYGM?
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 A02971-1 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 PYGM 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 is the PYGM band near 97 kDa?
Interpretation · The observed band near 97 kDa agrees with the 97.1 kDa predicted mass of canonical PYGM. Its listed modifications do not, by themselves, establish a visible shift or explain any small mass difference.
UniProt describes PYGM as a homodimer and as a homotetramer in enzymatically active phosphorylase A. If a high molecular weight band appears, test whether sample preparation preserves oligomers before assigning it to PYGM. Oligomerization alone does not establish that such a band will appear on a Western blot.
Check whether the antibody recognizes the region missing from isoform 2: canonical residues 82–169. A band below the approximately 97 kDa canonical band could be consistent with isoform 2, but the supplied features do not identify an observed lower band or rule out other explanations.
UniProt describes PYGM as a homodimer and as a homotetramer in enzymatically active phosphorylase A. If a high molecular weight band appears, test whether sample preparation preserves oligomers before assigning it to PYGM. Oligomerization alone does not establish that such a band will appear on a Western blot.
Check whether the antibody recognizes the region missing from isoform 2: canonical residues 82–169. A band below the approximately 97 kDa canonical band could be consistent with isoform 2, but the supplied features do not identify an observed lower band or rule out other explanations.
Boster reagents
PYGM 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-PYGM Antibody Picoband®
Cat # A02971-1
The catalog reports one anti-PYGM antibody for Western blotting, A02971-1. Its image shows a band near the expected 97 kDa in rat and mouse heart lysates; the supplied evidence does not show a human blot.
Which to pick: A02971-1 is the only listed option. It is catalogued as reactive with human, mouse, and rat; its supplied Western blot image uses rat and mouse heart lysates at 0.5 μg/mL antibody.
Source: BosterBio PYGM gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P11217.
- Human Protein Atlas. PYGM tissue expression.
- PMC6193451 — target-verified WB comparison
- PMC6994938 — target-verified WB comparison
- PMC8901937 — target-verified WB comparison