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ACTG2 / Actin, gamma-enteric smooth muscle · Western blot design guide
Design a Western Blot for ACTG2
Real validated ACTG2 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-ACTG2 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
ACTG2 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 | ~41.9 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Appendix (IHC candidate; verify WB) +4 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Acetylated + Methylated | |
| Caveat | Peptide-blocking control | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated ACTG2 Western Blot Protocols
The A05066 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | various cells (catalog A05066) |
| 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 | A05066 · 1:2000 (catalog A05066) |
| 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 ACTG2 Western Blot Band Size?
ACTG2 is predicted at 41.9 kDa; isoforms and listed modifications could affect migration, but no distinct band pattern or empirical size is established.
| Band near 41.9 kDa | consistent with the predicted ACTG2 monomer mass; confirm identity with antibody controls |
| Several bands near the monomer region | isoforms 1 and 2 are possible contributors, but their migration difference is unknown |
| Higher-mass band | may reflect incompletely dissociated actin assemblies |
| Weak band in a soluble lysate fraction | ACTG2 may remain in the cytoskeletal fraction |
💡Expected ACTG2 appearanceACTG2 has a predicted mass of 41.9 kDa, but no empirical band size is supplied; confirm a band near that size with antibody specificity and sample controls.
| UniProt predicted mass | places the ACTG2 monomer near 41.9 kDa |
| Isoforms 1 and 2 | could differ in size, but their masses and separation are unknown |
| N-acetylglutamate at residue 3 | is a listed modification without an established visible band shift |
| Tele-methylhistidine at residue 74 | has no established visible band shift |
| Methionine sulfoxide at residues 45 and 48 | has no established visible band shift |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | cytoskeletal ACTG2 may be poorly recovered by the extraction method | check the insoluble fraction and extraction conditions |
| Band higher than expected | actin assemblies may be incompletely dissociated | check denaturation and compare sample preparation conditions |
| Multiple bands | isoforms 1 and 2 are possible, but distinct migration is unproven | use antibody specificity controls before assigning band identities |
| Weak or no signal | the sample may contain little ACTG2 or yield poor cytoskeletal recovery | check sample expression, loading, and extraction |
| Fragments below expected size | sample degradation is possible; no fragment size is supplied | compare freshly prepared samples and use antibody specificity controls |
Experiment evidence
Western blot images showing ACTG2 band
A05066 · ACTG2
A05066 · ACTG2
A05066 · ACTG2
A05066-1 · ACTG2
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for ACTG2 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for ACTG2 in Western blot, you can use appendix tissue, which HPA rates as highly expressed.
Positive control: Appendix (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 appendix and no detection in adipose tissue, making tissue controls feasible.
HPA tissue expression evidence for ACTG2
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 | enterocytes - Microvilli | High | Protein (IHC) | HPA → |
| Colon | enterocytes - Microvilli | High | Protein (IHC) | HPA → |
| Duodenum | enterocytes - Microvilli | High | Protein (IHC) | HPA → |
| Rectum | enterocytes - Microvilli | High | Protein (IHC) | HPA → |
| Skin | fibrohistiocytic cells | 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 → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Caudate | glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ACTG2 Western Blot Tips
Deeper troubleshooting and optimisation questions for ACTG2, answered from its protein features.
How should ACTG2 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 ACTG2 isoform 2 produce a different band?
Isoforms · Isoform 2 lacks canonical residues 43–85, so it may migrate differently from isoform 1. No observed band position is supplied for either isoform; confirm band identity before assigning it.
Check whether the antibody epitope lies within canonical residues 43–85. That segment is missing from isoform 2, so an antibody targeting it may detect isoform 1 while missing isoform 2.
Check whether the antibody epitope lies within canonical residues 43–85. That segment is missing from isoform 2, so an antibody targeting it may detect isoform 1 while missing isoform 2.
Which N-terminal modifications are listed for ACTG2?
PTM · UniProt lists N-acetylcysteine at position 2 in an intermediate form and N-acetylglutamate at position 3 in ACTG2. These are UniProt coordinates; check the numbering convention when comparing antibody or paper descriptions. Their presence does not establish a visible band shift.
Does this guide establish induction of ACTG2?
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 ACTG2?
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 A05066 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.
What matters when quantifying ACTG2 by Western blot?
Quantitation · ACTG2 occurs as globular actin and polymerizes into filamentous actin in the cytoskeleton. Keep extraction conditions consistent across samples and assess whether ACTG2 recovery is comparable before interpreting band intensity as a change in abundance.
Should ACTG2 run at exactly 41.9 kDa?
Interpretation · 41.9 kDa is the predicted mass of canonical ACTG2. No observed band position is supplied. The listed modifications alone do not establish a visible shift or explain a difference between apparent and predicted mass.
UniProt lists methionine (R)-sulfoxide at positions 45 and 48. These are UniProt coordinates, and both residues lie in the segment missing from isoform 2. The entries alone do not establish when oxidation occurs or whether it changes band migration.
UniProt lists tele-methylhistidine at position 74, using UniProt numbering. Position 74 lies in the segment missing from isoform 2. The modification entry alone does not predict a separate Western blot band.
Consider the two listed isoforms first: isoform 2 lacks residues 43–85. Check the antibody epitope and compare bands with the 41.9 kDa canonical prediction. The supplied features do not establish observed band positions or show that any listed modification creates a separate band.
UniProt lists methionine (R)-sulfoxide at positions 45 and 48. These are UniProt coordinates, and both residues lie in the segment missing from isoform 2. The entries alone do not establish when oxidation occurs or whether it changes band migration.
UniProt lists tele-methylhistidine at position 74, using UniProt numbering. Position 74 lies in the segment missing from isoform 2. The modification entry alone does not predict a separate Western blot band.
Consider the two listed isoforms first: isoform 2 lacks residues 43–85. Check the antibody epitope and compare bands with the 41.9 kDa canonical prediction. The supplied features do not establish observed band positions or show that any listed modification creates a separate band.
Boster reagents
ACTG2 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-Actin alpha3 ACTG2 Antibody
Cat # A05066
Real WB data
Anti-Actin-Alpha 3/ Gamma 2 (E2) ACTG2 Antibody
Cat # A05066-1
Two the supplier anti-ACTG2 antibodies, A05066 and A05066-1, list human, mouse, and rat reactivity and each has a WB image. A05066’s caption describes various cells without naming them; A05066-1’s caption names five whole-cell lysates and a 1:500 dilution.
Which to pick: Both list the same species reactivity and have WB images. Choose A05066-1 if its documented whole-cell lysate conditions are useful for your design; choose A05066 if its reported 1:2000 dilution better matches your planned starting conditions.
Source: BosterBio ACTG2 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P63267.
- Human Protein Atlas. ACTG2 tissue expression.
- PMC11378013 — target-verified WB comparison
- PMC7455133 — target-verified WB comparison
- PMC7835941 — target-verified WB comparison