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- Table of Contents
AGO4 / Protein argonaute-4 · Western blot design guide
Design a Western Blot for AGO4
Real validated AGO4 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-AGO4 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
AGO4 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 | ~97.1 kDa | |
| Observed band | ~100 kDa | |
| Gel | 10% (catalog PB9979) | |
| Positive control | Fallopian tube (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 | Ubl conjugation | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated AGO4 Western Blot Protocols
The PB9979 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Hela, human 293T, human Jurkat (catalog PB9979) |
| Gel % | 10% (catalog PB9979) |
| Load | 30 ug; reducing conditions (catalog PB9979) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog PB9979) |
| Membrane | nitrocellulose membrane (catalog PB9979) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog PB9979) |
| Primary antibody | PB9979 · 0.5 μg/mL (catalog PB9979) |
| Primary incubation | overnight at 4°C (catalog PB9979) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog PB9979) |
| Secondary incubation | 1.5 hour at RT (catalog PB9979) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog PB9979) |
| Detection | ECL (catalog PB9979) |
Section 2
What Is the Expected AGO4 Western Blot Band Size?
AGO4 is predicted at 97.1 kDa and observed at approximately 100 kDa; the cause of the difference is not established by the supplied features.
| Band at approximately 100 kDa | Matches the reported AGO4 band in whole-cell lysate; confirm identity with controls |
| Band near 97.1 kDa | Near AGO4's predicted mass |
| Single sharp band near 100 kDa | Consistent with the reported band and one annotated isoform |
| Band present in whole-cell lysate | Consistent with AGO4's cytoplasmic location |
💡Expected AGO4 appearanceAGO4 has a predicted mass of 97.1 kDa and a reported band at approximately 100 kDa in whole-cell lysate; confirm band identity with appropriate controls.
| UniProt predicted mass | Places AGO4 at 97.1 kDa before considering its observed migration |
| Predicted molecular weight in daltons | Gives the same predicted size as 97,097 Da |
| Predicted full-length size | Provides a 97.1 kDa reference for the intact protein |
| Predicted mass versus observed migration | The predicted 97.1 kDa protein has a reported band at approximately 100 kDa; the cause of the difference is not established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | AGO4 signal may be below detection in the tested sample | Check loading and antibody performance with a positive-control lysate |
| Band higher than expected | No supplied feature establishes a larger AGO4 species | Compare with the approximately 100 kDa reference band and verify identity by AGO4 depletion |
| Band lower than expected | No annotated processing explains a smaller band | Check sample integrity and verify identity by AGO4 depletion |
| Multiple bands | Only one isoform is annotated; additional bands have no established explanation here | Use AGO4 depletion or an independent antibody to identify the specific band |
| Weak or no signal | AGO4 signal may be below the assay's detection limit | Check loading, transfer, and antibody performance with a positive-control lysate |
| Fragments below expected size | Sample degradation is possible, but no fragment size is supplied | Prepare fresh lysate with protease inhibitors and verify band identity |
Experiment evidence
Western blot images showing AGO4 band
PB9979 · AGO4
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for AGO4 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for AGO4 in Western blot, you can use fallopian tube lysate, which shows high expression in HPA.
Positive control: Fallopian tube (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: AGO4 is cytoplasmic, and HPA reports a not-detected tissue for a negative control.
HPA tissue expression evidence for AGO4
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 |
|---|---|---|---|---|
| Fallopian tube | ciliated cells (cell body) | High | Protein (IHC) | HPA → |
| Kidney | cells in glomeruli | High | Protein (IHC) | HPA → |
| Liver | cholangiocytes | High | Protein (IHC) | HPA → |
| Pancreas | exocrine glandular cells | High | Protein (IHC) | HPA → |
| Prostate | glandular 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 → |
| Appendix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
| Caudate | glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced AGO4 Western Blot Tips
Deeper troubleshooting and optimisation questions for AGO4, answered from its protein features.
How should AGO4 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 AGO4 isoforms explain multiple bands?
Isoforms · The supplied record lists one isoform and no alternative sequence. It provides no isoform-based explanation for multiple bands.
Could a modification shift the AGO4 band?
PTM · Ubl conjugation is listed as a keyword, but no modified residue or site is supplied. That annotation alone does not establish a visible band shift.
Does this guide establish induction of AGO4?
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.
What transfer method to use for AGO4 Western blot?
Transfer · AGO4 is predicted at 97.1 kDa and observed near 100 kDa. The supplied features do not specify a transfer method; verify that your chosen conditions transfer proteins around 100 kDa efficiently.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the PB9979 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 AGO4 localization affect quantitation?
Quantitation · AGO4 is annotated in the cytoplasm and P-bodies. Compare the ~100-kDa band across samples prepared the same way, especially when using subcellular fractions.
Why might AGO4 appear near 100 kDa instead of 97.1 kDa?
Interpretation · The supplied empirical band is ~100 kDa, close to the predicted 97.1 kDa. The listed features do not establish what causes the difference in apparent mass.
The supplied record lists one isoform, no signal peptide or propeptide, and no glycosylation sites. These features do not explain extra bands. Check whether the ~100-kDa band is reproducible before assigning an identity to other bands.
The supplied record lists one isoform, no signal peptide or propeptide, and no glycosylation sites. These features do not explain extra bands. Check whether the ~100-kDa band is reproducible before assigning an identity to other bands.
Boster reagents
AGO4 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-Argonaute 4/AGO4 Antibody Picoband®
Cat # PB9979
PB9979 is a rabbit polyclonal anti-AGO4 antibody listed for human, mouse, and rat. Its Western blot image shows a band near 100 kDa in human HeLa, 293T, and Jurkat whole cell lysates; the expected size is 97 kDa. Mouse and rat blot examples are not supplied.
Which to pick: PB9979 is the only listed option. Its Western blot image documents human cell lysates at 30 µg per lane, using 0.5 µg/mL primary antibody. For mouse or rat samples, the stated reactivity lacks a supplied blot example.
Source: BosterBio AGO4 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9HCK5.
- Human Protein Atlas. AGO4 tissue expression.
- PMC4971500 — target-verified WB comparison
- PMC6620710 — target-verified WB comparison