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PRKAG2 / 5'-AMP-activated protein kinase subunit gamma-2 · Western blot design guide
Design a Western Blot for PRKAG2
Source-linked PRKAG2 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-PRKAG2 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
PRKAG2 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 | ~63.1 kDa | |
| Observed band | ~63 kDa | |
| Gel | 5–20% (catalog A02758-3) | |
| 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 | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 3 isoform(s) |
Section 1
Source-Linked PRKAG2 Western Blot Protocol Options
The A02758-3 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human 293T, human Hela, human placenta, human U-87MG, human A431, rat heart, mouse NIH/3T3 (catalog A02758-3) |
| Gel % | 5–20% (catalog A02758-3) |
| Load | 30 ug; reducing conditions (catalog A02758-3) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A02758-3) |
| Membrane | nitrocellulose membrane (catalog A02758-3) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A02758-3) |
| Primary antibody | A02758-3 · 0.5 μg/mL (catalog A02758-3) |
| Primary incubation | overnight at 4°C (catalog A02758-3) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A02758-3) |
| Secondary incubation | 1.5 hour at RT (catalog A02758-3) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A02758-3) |
| Detection | ECL (catalog A02758-3) |
Section 2
What Is the Expected PRKAG2 Western Blot Band Size?
PRKAG2 is predicted at 63.1 kDa and observed at ~63 kDa; isoforms and phosphosites are documented, but distinct migration effects are not established.
| Band at ~63 kDa | Matches the empirical PRKAG2 band and its 63.1 kDa predicted mass |
| Several bands near ~63 kDa | Isoforms A, B and C could contribute, but their migration is unknown |
| Doublet near ~63 kDa | Phosphorylation could affect migration, but its visible effect is unestablished |
| Single sharp band near ~63 kDa | The listed isoforms and phosphosites need not produce resolvable bands |
💡Expected PRKAG2 appearancePRKAG2 is predicted at 63.1 kDa and observed at ~63 kDa in reducing whole-cell blots; confirm band identity with an appropriate positive sample and antibody specificity control.
| Predicted PRKAG2 mass | 63.1 kDa predicts a band near the observed ~63 kDa |
| Isoforms A, B and C | May differ in size, but their masses and migration are not supplied |
| Phosphoserine at Ser65 | Could affect migration; no visible shift is established |
| Phosphoserines at Ser71 and Ser73 | Could affect migration; no visible shift is established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | PRKAG2 signal may be below detection or the assay may have failed | Compare with a positive lysate and check loading, transfer and antibody performance |
| Band higher than expected | An isoform or phosphorylation may affect migration, but neither explains a specific shift here | Compare with the ~63 kDa control band and verify identity with a second antibody |
| Band lower than expected | An isoform may differ in size, but its mass is unknown | Compare isoform expression and verify the band with a second antibody |
| Multiple bands | Isoforms or phosphorylation could contribute; band identities are unconfirmed | Compare with a positive control and use an independent antibody to identify PRKAG2 bands |
| Weak or no signal | PRKAG2 abundance or assay sensitivity may be insufficient | Check loading and transfer, then compare with a positive lysate |
Experiment evidence
Western blot images showing PRKAG2 band
A02758-3 · PRKAG2
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for PRKAG2 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for PRKAG2 in Western blot, you can use appendix tissue, which has high HPA expression.
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 reports expression across the listed tissues, so use siRNA knockdown or a KO line for a clean negative control.
HPA tissue expression evidence for PRKAG2
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 → |
| Bone marrow | hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cerebellum | cells in molecular layer | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Skeletal muscle | myocytes | Low | Protein (IHC) | HPA → |
| Adipose tissue | adipocytes | Medium | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Medium | Protein (IHC) | HPA → |
| Caudate | glial cells | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | glial cells | Medium | Protein (IHC) | HPA → |
Section 3
Advanced PRKAG2 Western Blot Tips
Deeper troubleshooting and optimisation questions for PRKAG2, answered from its protein features.
How should PRKAG2 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.
How might PRKAG2 isoforms affect band interpretation?
Isoforms · UniProt lists isoforms A, B and C. Relative to the canonical sequence, B lacks residues 1–241 and C lacks residues 1–44. These differences can affect band position and antibody recognition; the supplied evidence does not give their observed band sizes.
Check where the epitope falls in the canonical sequence. An epitope within residues 1–241 is absent from isoform B; one within residues 1–44 is absent from both B and C. Interpret missing isoform bands in light of that coverage.
Check where the epitope falls in the canonical sequence. An epitope within residues 1–241 is absent from isoform B; one within residues 1–44 is absent from both B and C. Interpret missing isoform bands in light of that coverage.
Which PRKAG2 phosphorylation sites matter for phospho-specific detection?
PTM · In UniProt canonical numbering, phosphoserines occur at 65, 71, 73, 90, 138, 143, 161, 162 and 196; phosphothreonine occurs at 165. Match the antibody’s stated site and numbering convention to these coordinates.
PRKAG2 has ten listed phosphorylation sites, but their presence alone does not demonstrate a visible shift. Compare the band with the approximately 63 kDa observation and consider the listed isoforms before assigning a shifted band to phosphorylation.
PRKAG2 has ten listed phosphorylation sites, but their presence alone does not demonstrate a visible shift. Compare the band with the approximately 63 kDa observation and consider the listed isoforms before assigning a shifted band to phosphorylation.
Does this guide establish induction of PRKAG2?
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 PRKAG2?
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 A02758-3 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 can PRKAG2 bands be quantified consistently?
Quantitation · Define which isoform or bands the antibody detects before comparing signal across samples. Because isoforms B and C have different N-terminal deletions, an antibody’s epitope can change which forms contribute to the measured signal.
Why is the PRKAG2 band near 63 kDa?
Interpretation · The predicted mass is 63.1 kDa, and the supplied observed band is approximately 63 kDa. The listed features do not establish a detectable mass shift.
Boster reagents
PRKAG2 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-PRKAG2 Antibody Picoband®
Cat # A02758-3
A02758-3 is listed for human, mouse, and rat PRKAG2. Its Western blot image shows an approximately 63 kDa band in the specified human cell and placenta, rat heart, and mouse NIH/3T3 lysates. No publication evidence is supplied.
Which to pick: A02758-3 is the only listed option and has a Western blot image. For a comparable sample, use the caption’s tested conditions as a starting point: 30 µg reducing lysate and 0.5 µg/mL primary antibody.
Source: BosterBio PRKAG2 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9UGJ0.
- Human Protein Atlas. PRKAG2 tissue expression.
- PMC10950609 — target-verified WB comparison
- PMC10629191 — target-verified WB comparison