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RPS6KA1 / Ribosomal protein S6 kinase alpha-1 · Western blot design guide
Design a Western Blot for RPS6KA1
Source-linked RPS6KA1 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-RPS6KA1 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
RPS6KA1 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 | ~82.7 kDa | |
| Gel | 8–10% (standard starting point) | |
| Positive control | Appendix (IHC candidate; verify WB) +4 more | |
| Negative control | Ovary (IHC candidate; verify WB) |
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 | 4 isoform(s) |
Section 1
Source-Linked RPS6KA1 Western Blot Protocol Options
The M01058-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | MCF7 cell lysate (catalog M01058-2) |
| Gel % | 8–10% (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 | M01058-2; use the WB datasheet starting dilution (standard starting point) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | Species-matched HRP conjugate at validated dilution (standard starting point) |
| Secondary incubation | 1 h at room temperature (standard starting point) |
| 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 RPS6KA1 Western Blot Band Size?
RPS6KA1 is predicted at 82.7 kDa; isoforms and phosphorylation could affect migration, but no empirical band size or visible shift is supplied.
| Band near 82.7 kDa | consistent with the predicted full-length RPS6KA1 mass, pending band-identity controls |
| Bands at different sizes | could reflect isoforms 1, 2, 3, and 4; their migration is not supplied |
| Close doublet near 82.7 kDa | could reflect phosphorylation, though a visible mobility shift is not established |
| One band without resolved variants | the listed isoforms and phosphorylation sites need not produce separate visible bands |
💡Expected RPS6KA1 appearanceUniProt predicts 82.7 kDa for RPS6KA1, but no empirical band size is supplied; confirm any candidate band with ordinary identity controls.
| UniProt predicted mass | 82.7 kDa is the reference mass, not a measured blot position |
| Isoform 1 | its individual mass and migration are not supplied |
| Isoform 2 | alternative splicing may alter size, but its mass is not supplied |
| Isoform 3 | alternative splicing may alter size, but its mass is not supplied |
| Isoform 4 | alternative splicing may alter size, but its mass is not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | nuclear or cytoplasmic RPS6KA1 may be underrecovered during extraction | compare whole-cell lysate with nuclear and cytoplasmic fractions using a positive control |
| Band higher than expected | phosphorylation may affect mobility, but the band identity is unverified | compare phosphatase-treated lysate and confirm with an independent antibody |
| Band lower than expected | an alternative isoform is possible, but isoform masses are not supplied | check isoform expression and confirm with an antibody to another epitope |
| Multiple bands | isoforms or phosphorylation could contribute; distinct migration is unproven | compare phosphatase-treated samples and verify candidate bands with an independent antibody |
| Weak or no signal | extraction may underrecover RPS6KA1 from its nuclear or cytoplasmic location | check fraction recovery and run a positive-control lysate |
Experiment evidence
Western blot images showing RPS6KA1 band
M01058-2 · RPS6KA1
PA2052 · RPS6KA1
PA2052 · RPS6KA1
P01058-1 · RPS6KA1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for RPS6KA1 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for RPS6KA1 in Western blot, you can use appendix tissue, which has high HPA expression.
Positive control: Appendix (IHC candidate; verify WB)
Negative control: Ovary (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: Appendix and HPA-undetected ovary provide a feasible tissue contrast, though Western blot sensitivity may differ.
HPA tissue expression evidence for RPS6KA1
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 → |
| Cervix | glandular cells | High | Protein (IHC) | HPA → |
| Colon | glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | glandular cells | High | Protein (IHC) | HPA → |
| Endometrium | glandular cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Ovary | ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
| Adipose tissue | adipocytes | Low | Protein (IHC) | HPA → |
| Cerebellum | cells in granular layer | Low | Protein (IHC) | HPA → |
| Liver | cholangiocytes | Low | Protein (IHC) | HPA → |
| Skeletal muscle | myocytes | Low | Protein (IHC) | HPA → |
Section 3
Advanced RPS6KA1 Western Blot Tips
Deeper troubleshooting and optimisation questions for RPS6KA1, answered from its protein features.
How should RPS6KA1 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.
Which RPS6KA1 isoforms could affect band interpretation?
Isoforms · Four isoforms are listed. Isoform 3 lacks canonical residues 1–92; isoforms 2 and 4 replace N-terminal sequences. These differences could affect apparent size or antibody recognition, but the supplied features do not establish where any isoform migrates.
Check whether the antibody epitope overlaps the altered N terminus. Canonical residues 1–92 are missing in isoform 3; residues 1–36 are replaced in isoform 2, and 1–35 in isoform 4. An epitope in those regions may detect isoforms differently.
Check whether the antibody epitope overlaps the altered N terminus. Canonical residues 1–92 are missing in isoform 3; residues 1–36 are replaced in isoform 2, and 1–35 in isoform 4. An epitope in those regions may detect isoforms differently.
Which phosphorylation sites matter for phospho-specific RPS6KA1 blots?
PTM · The supplied UniProt coordinates are Ser54, Ser221, Ser307, Thr359, Ser363, Ser369, Ser380, Thr573 and Ser732. Ser221 is phosphorylated by PDPK1; Ser380 is phosphorylated by autocatalysis. Match the antibody's stated numbering convention to these UniProt coordinates before interpreting a site-specific signal.
How should mitogenic stimulation affect RPS6KA1 interpretation?
Induction · RPS6KA1 forms a complex with MAPK1/ERK2 or MAPK3/ERK1 in quiescent cells and transiently dissociates following mitogenic stimulation. Record stimulation conditions when comparing samples; this feature alone does not establish a change in total RPS6KA1 or phosphorylation at a particular site.
How should transfer be checked for RPS6KA1?
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 M01058-2 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 RPS6KA1 be quantified across samples?
Quantitation · RPS6KA1 is listed in both nucleus and cytoplasm, so compare matched sample fractions. Assess total RPS6KA1 separately from any site-specific phospho signal: a change in phospho signal alone does not establish a change in protein abundance.
Should RPS6KA1 migrate at exactly 82.7 kDa?
Interpretation · 82.7 kDa is the predicted mass; no observed band position is supplied. The listed phosphorylation sites and alternative sequences do not, by themselves, establish a visible shift or explain a difference from 82.7 kDa.
Check whether the antibody recognizes the altered N termini of isoforms 2, 3 and 4, then compare total and site-specific phospho signals. The listed isoforms and nine phosphorylation sites are possibilities to investigate; the supplied features do not identify an unexpected band or prove that phosphorylation shifts its migration.
Check whether the antibody recognizes the altered N termini of isoforms 2, 3 and 4, then compare total and site-specific phospho signals. The listed isoforms and nine phosphorylation sites are possibilities to investigate; the supplied features do not identify an unexpected band or prove that phosphorylation shifts its migration.
Boster reagents
RPS6KA1 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-RSK1 p90 Rabbit Monoclonal Antibody
Cat # M01058-2
Real WB data
Anti-RSK1 p90/RPS6KA1 Antibody Picoband®
Cat # PA2052
Real WB data
Anti-Phospho-RSK1 (S380) RPS6KA1 Rabbit Monoclonal Antibody
Cat # P01058-1
Three the supplier antibodies are listed for RPS6KA1 Western blots: two against RSK1 p90 and one against phospho-RSK1 S380. Each has a WB image. The supplied captions show human lysates for all three and rat PC-12 lysate for the phospho antibody; none shows mouse samples.
Which to pick: Choose M01058-2 for the shown MCF-7 context, or PA2052 for its broader reported reactivity and images across six human cell lines. Choose P01058-1 to detect phospho-S380; its image includes human and rat lysates with an approximately 83 kDa band. Mouse reactivity is listed but not shown.
Source: BosterBio RPS6KA1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q15418.
- Human Protein Atlas. RPS6KA1 tissue expression.
- PMC7646838 — target-verified WB comparison
- PMC11743407 — target-verified WB comparison
- PMC10400424 — target-verified WB comparison