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- Table of Contents
PRKACA / cAMP-dependent protein kinase catalytic subunit alpha · Western blot design guide
Design a Western Blot for PRKACA
Real validated PRKACA Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-PRKACA 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
PRKACA 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 | ~40.6 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Cerebral cortex (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 Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated PRKACA Western Blot Protocols
The A00653 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | Target-positive lysate and matched negative control (standard starting point) |
| Gel % | 12–15% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Standard semi-dry transfer; verify efficiency (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | A00653; use the WB datasheet starting dilution (standard starting point) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | 1:5000 (catalog A00653) |
| Secondary incubation | 1hour (catalog A00653) |
| Wash | 3 × 5 min in TBST (standard starting point) |
| Detection | ECL; bracket exposures to avoid saturation (standard starting point) |
Section 2
What Is the Expected PRKACA Western Blot Band Size?
PRKACA is predicted at 40.6 kDa; isoforms and modifications could affect migration, but no empirical band or feature-specific shift is established.
| Band near 40.6 kDa | Consistent with the predicted PRKACA size; confirm identity with antibody controls |
| Two nearby bands | Could reflect isoforms 1 and 2 or modified PRKACA; their migration is not established |
| Slightly shifted band | Could reflect a modified PRKACA form; its migration is not established |
| Weak band in one cell fraction | PRKACA occurs in several cellular compartments |
💡Expected PRKACA appearanceUniProt predicts 40.6 kDa for PRKACA; no empirical band is supplied, and isoforms or modifications have no demonstrated migration effect here, so confirm any candidate band with specificity controls.
| UniProt predicted mass | Places the reference size at 40.6 kDa |
| Alternative splicing | Produces isoforms 1 and 2; their relative sizes are not supplied |
| Isoform 1 | Has no supplied isoform-specific mass or migration |
| Isoform 2 | Has no supplied isoform-specific mass or migration |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Signal may be below detection or lost during extraction | Check loading, extraction, and a positive-control lysate |
| Band higher than expected | Isoform or modification effects are possible but unverified | Confirm identity with a second antibody or PRKACA depletion |
| Band lower than expected | An isoform size difference is possible but unquantified | Confirm identity with PRKACA depletion or an isoform-specific control |
| Multiple bands | Isoforms or modified forms are possible; distinct migration is unproven | Compare bands after PRKACA depletion and with a second antibody |
| Weak or no signal | PRKACA is distributed among cellular compartments | Check relevant cell fractions and loading controls |
Experiment evidence
Western blot images showing PRKACA band
A00653 · PRKACA
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for PRKACA Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for PRKACA in Western blot, you can use cerebral cortex lysate, which has high HPA expression.
Positive control: Cerebral cortex (IHC candidate; verify WB)
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: No HPA tissue is listed as not detected, so use siRNA knockdown or a KO line for a clean negative control.
HPA tissue expression evidence for PRKACA
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 |
|---|---|---|---|---|
| Cerebral cortex | neuronal cells | High | Protein (IHC) | HPA → |
| Colon | peripheral nerve/ganglion | High | Protein (IHC) | HPA → |
| Soft tissue | chondrocytes | High | Protein (IHC) | HPA → |
| Testis | cells in seminiferous ducts | High | Protein (IHC) | HPA → |
| Adipose tissue | adipocytes | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Bone marrow | hematopoietic cells | Low | Protein (IHC) | HPA → |
| Breast | adipocytes | Low | Protein (IHC) | HPA → |
| Duodenum | glandular cells | Low | Protein (IHC) | HPA → |
| Esophagus | squamous epithelial cells | Low | Protein (IHC) | HPA → |
| Gallbladder | glandular cells | Low | Protein (IHC) | HPA → |
Section 3
Advanced PRKACA Western Blot Tips
Deeper troubleshooting and optimisation questions for PRKACA, answered from its protein features.
Where should the main PRKACA band appear?
Band shift · The predicted mass is 40.6 kDa. No empirical band position is supplied, so use 40.6 kDa as a reference rather than an expected apparent mass. The listed modifications alone do not establish a visible shift.
Could PRKACA isoforms produce nearby bands?
Isoforms · UniProt lists two isoforms. Isoform 2 replaces canonical residues 1–15 (MGNAAAAKKGSEQES) with MASNSSD, shortening the sequence by eight residues. Whether both isoforms are expressed or resolved as separate bands in your sample is unknown.
Which PRKACA modifications matter when interpreting bands?
PTM · In canonical UniProt numbering, Asn3 is deamidated; Ser11 is phosphorylated by autocatalysis; Thr49, Ser140, Thr196, Tyr331, and Ser339 are phosphorylated; and Thr198 is phosphorylated by PDPK1. These features do not establish that any particular band shift will be visible. The altered N terminus of isoform 2 means its residue numbering may differ.
Does cAMP stimulation imply more PRKACA protein?
Induction · The supplied holoenzyme feature says cAMP causes dissociation of regulatory and catalytic subunits. That supports a change in complex state, but does not establish increased PRKACA abundance or a stronger Western blot band after stimulation.
How should transfer be checked for PRKACA?
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 A00653 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 PRKACA be quantified across cell fractions?
Quantitation · PRKACA is listed in the cytoplasm, nucleus, membranes, and mitochondrion. Compare like fractions across samples and account for fraction recovery when interpreting band intensity; a change in one fraction alone need not reflect a change in total PRKACA.
How should unexpected PRKACA bands be interpreted?
Interpretation · Check the 40.6 kDa predicted mass, the two isoforms, and the listed modifications as possible leads. The features do not assign an apparent mass to either isoform or demonstrate a modification-dependent shift, so band position alone cannot identify an unexpected band.
Boster reagents
PRKACA 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-PKA alpha/ beta/ gamma cat PRKACA Antibody
Cat # A00653
A00653 is catalogued for human, mouse, pig, and rat and has a Western blot image using 22RV1, HeLa, and COLO205 lysates. The supplied evidence does not show WB testing in mouse, pig, or rat samples.
Which to pick: A00653 is the only listed antibody. Its WB image uses 22RV1, HeLa, and COLO205 lysates with the primary antibody diluted 1:1000; consider whether those conditions fit your experiment.
Source: BosterBio PRKACA gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P17612.
- Human Protein Atlas. PRKACA tissue expression.
- PMC7895437 — target-verified WB comparison
- PMC12267505 — target-verified WB comparison
- PMC12911764 — target-verified WB comparison