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RGS16 / Regulator of G-protein signaling 16 · Western blot design guide
Design a Western Blot for RGS16
Source-linked RGS16 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-RGS16 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
RGS16 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 | ~22.7 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Bronchus (total-target IHC; phospho state unverified) +4 more | |
| Negative control | Adipose tissue (total-target IHC; phospho state unverified) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated | |
| Caveat | Activation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Source-Linked RGS16 Western Blot Protocol Options
The P04881 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | lysates from COS7 cells treated with heat shock, (catalog P04881) |
| Gel % | 12–15% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Short semi-dry transfer; verify retention (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | the phospho peptide (catalog P04881) |
| Primary antibody | P04881; 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 RGS16 Western Blot Band Size?
RGS16 has a predicted mass of 22.7 kDa, with no empirical band size supplied; phosphorylation and lipid anchoring have no demonstrated migration effect here.
| Band near 22.7 kDa | Consistent with the predicted RGS16 mass; identity requires controls |
| Band detected by anti-phospho-Tyr168 antibody | Consistent with RGS16 phosphorylated at Tyr168, not a measurement of total RGS16 |
| Little or no band in soluble lysate | Membrane-associated RGS16 may remain in the insoluble fraction |
| Band lost after phosphopeptide blocking | Supports recognition of the phospho-Tyr168 epitope |
💡Expected RGS16 appearanceUniProt predicts 22.7 kDa for RGS16, but no empirical band size is supplied; the lead antibody recognizes phospho-Tyr168, so confirm band identity with phosphopeptide blocking and an appropriate total-RGS16 control.
| UniProt predicted mass | Places the expected full-length band near 22.7 kDa; migration is unverified |
| Phosphotyrosine at Tyr168 by EGFR | Defines the lead antibody's target state; no visible size shift is established |
| Phosphotyrosine at Tyr177 | May alter phosphorylation state; no visible size shift is established |
| Membrane lipid anchor | May affect recovery in lysate; no apparent-size effect is established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Membrane-associated RGS16 may be poorly recovered | Check the membrane fraction and lysate preparation |
| Band higher than expected | Its identity and cause of migration are unestablished | Check phosphopeptide blocking and compare with a total-RGS16 antibody |
| Band lower than expected | Possible degradation or unrelated antibody binding | Check sample integrity and confirm identity with an independent antibody |
| Multiple bands | Band identities are unestablished | Use phosphopeptide blocking and a total-RGS16 antibody to identify the relevant band |
| Weak or no signal | The phospho-Tyr168 antibody requires its phosphorylated epitope | Include a phospho-Tyr168 positive control and check total RGS16 |
| Fragments below expected size | Possible sample degradation | Prepare fresh lysate with protease inhibitors and verify with an independent antibody |
Experiment evidence
Western blot images showing RGS16 band
P04881 · RGS16
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for RGS16 Western blot
🧪HPA-IHC candidate guidance (verify in WB): HPA tissue expression does not establish phosphorylation at the selected antibody epitope. Verify activation-state controls for the phospho-specific lead. HPA-IHC candidate guidance (verify in WB): For positive controls for RGS16 in Western blot, you can use bronchus tissue.
Positive control: Bronchus (total-target IHC; phospho state unverified)
Negative control: Adipose tissue (total-target IHC; phospho state unverified)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: RGS16 is membrane-associated, so membrane enrichment may help if whole-tissue lysate gives a weak signal.
HPA tissue expression evidence for RGS16
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 |
|---|---|---|---|---|
| Bronchus | respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
| Duodenum | glandular cells | Medium | Protein (IHC) | HPA → |
| Kidney | cells in tubules | Medium | Protein (IHC) | HPA → |
| Parathyroid gland | glandular cells | Medium | Protein (IHC) | HPA → |
| Skeletal muscle | myocytes | Medium | 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 → |
| Appendix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
| Caudate | glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced RGS16 Western Blot Tips
Deeper troubleshooting and optimisation questions for RGS16, answered from its protein features.
How should RGS16 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 RGS16 isoforms explain multiple bands?
Isoforms · The supplied record lists one isoform and no alternative sequence. It therefore provides no isoform-based explanation for multiple bands.
Which RGS16 phosphorylation sites should I consider?
PTM · UniProt lists phosphotyrosine at positions 168 and 177; position 168 is attributed to EGFR. These are UniProt coordinates, which may differ from paper or antibody numbering. Their presence does not establish a visible band shift.
Does this guide establish induction of RGS16?
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 RGS16?
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 P04881 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 I quantify RGS16 across samples?
Quantitation · Use comparable sample fractions and distinguish total RGS16 measurements from measurements of phosphorylation at UniProt positions 168 or 177. The membrane and lipid-anchor annotations make consistent recovery especially relevant. The features alone do not show that phosphorylation changes total RGS16 abundance.
Should RGS16 run at exactly 22.7 kDa?
Interpretation · 22.7 kDa is the predicted mass; no observed band position is supplied. Use it as a reference when assessing bands, but do not assign a band solely by its apparent mass. The listed modifications do not establish a visible shift.
EGFR is listed as phosphorylating RGS16 at UniProt position 168. If comparing conditions that alter EGFR activity, consider whether the assay detects total RGS16 or phosphorylation at that site. The supplied features do not establish how the band intensity responds to any particular treatment.
RGS16 is annotated as membrane-associated through a lipid anchor. When comparing samples, keep fraction collection and extraction consistent, and consider whether RGS16 could be present in a membrane fraction that was omitted.
Compare its position with the predicted 22.7 kDa mass and check sample fractionation and assay specificity. The record lists one isoform and phosphotyrosines at UniProt positions 168 and 177, but supplies no observed band position or evidence that either modification causes a visible shift.
EGFR is listed as phosphorylating RGS16 at UniProt position 168. If comparing conditions that alter EGFR activity, consider whether the assay detects total RGS16 or phosphorylation at that site. The supplied features do not establish how the band intensity responds to any particular treatment.
RGS16 is annotated as membrane-associated through a lipid anchor. When comparing samples, keep fraction collection and extraction consistent, and consider whether RGS16 could be present in a membrane fraction that was omitted.
Compare its position with the predicted 22.7 kDa mass and check sample fractionation and assay specificity. The record lists one isoform and phosphotyrosines at UniProt positions 168 and 177, but supplies no observed band position or evidence that either modification causes a visible shift.
Boster reagents
RGS16 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-Phospho RGS16 (Tyr168) Antibody
Cat # P04881
The listed antibody targets RGS16 phosphorylated at Tyr168. Its Western blot image shows heat-shocked COS7 lysate and a phosphopeptide-blocked lane. This evidence is limited to the reported conditions and does not establish detection of total RGS16.
Which to pick: Only P04881 is listed. It reports human, monkey, mouse, and rat reactivity, with a Western blot image from heat-shocked COS7 cells. Choose it when phosphorylated Tyr168 is the intended readout; the image does not establish Western blot performance in every listed species.
Source: BosterBio RGS16 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O15492.
- Human Protein Atlas. RGS16 tissue expression.
- PMC9439696 — target-verified WB comparison
- PMC12102885 — target-verified WB comparison
- PMC7366748 — target-verified WB comparison