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CHAC1 / Glutathione-specific gamma-glutamylcyclotransferase 1 · Western blot design guide
Design a Western Blot for CHAC1
Real validated CHAC1 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CHAC1 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
CHAC1 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 | ~24.4 kDa | |
| Observed band | ~40 kDa | |
| Gel | 12% (catalog A09017-2) | |
| Positive control | Appendix (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 | — | |
| Caveat | Band identity controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated CHAC1 Western Blot Protocols
The A09017-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human A549, human Hela, human U251 (catalog A09017-2) |
| Gel % | 12% (catalog A09017-2) |
| Load | 30 ug; reducing conditions (catalog A09017-2) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A09017-2) |
| Membrane | nitrocellulose membrane (catalog A09017-2) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A09017-2) |
| Primary antibody | A09017-2 · 1:1000 (catalog A09017-2) |
| Primary incubation | overnight at 4°C (catalog A09017-2) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A09017-2) |
| Secondary incubation | 1.5 hour at RT (catalog A09017-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A09017-2) |
| Detection | ECL (catalog A09017-2) |
Section 2
What Is the Expected CHAC1 Western Blot Band Size?
CHAC1 is predicted at 24.4 kDa, while antibody QC reports approximately 40 kDa; the cause of the difference is not established.
| Band near 40 kDa | Empirical CHAC1 band in reducing whole-cell lysates; confirm identity with controls |
| Band near 24.4 kDa | Near the UniProt predicted mass; identity requires confirmation |
| Several bands at different positions | Isoforms 1 and 2 are possible contributors, but distinct migration is unverified |
| Band in a cytosolic fraction | Consistent with CHAC1 cytosolic localization |
💡Expected CHAC1 appearanceUniProt predicts 24.4 kDa, while antibody QC shows a band near 40 kDa in reducing whole-cell lysates; its identity and the reason for the difference require ordinary controls.
| UniProt predicted mass | 24.4 kDa is the sequence-based reference, not demonstrated migration |
| Isoform 1 | Its individual apparent mass is not supplied |
| Isoform 2 | Its individual apparent mass is not supplied |
| Alternative splicing | Could alter mass, but distinct migration of isoforms 1 and 2 is unverified |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | The observed band is near 40 kDa versus a 24.4 kDa prediction; the cause is not established | Confirm identity with CHAC1 depletion and an independent antibody |
| Band lower than expected | A band near the predicted mass may represent CHAC1 or an unrelated signal | Check whether it decreases after CHAC1 depletion |
| Multiple bands | Isoforms 1 and 2 exist, but their migration is not established | Test which bands respond to CHAC1 depletion |
| No band in lysate | Fraction preparation may exclude cytosolic or trans-Golgi CHAC1 | Check the fraction collected and probe a whole-cell lysate control |
| Weak or no signal | Target abundance or detection may be insufficient | Check sample loading and use a CHAC1-positive lysate control |
Experiment evidence
Western blot images showing CHAC1 band
A09017-2 · CHAC1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CHAC1 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for CHAC1 in Western blot, you can use appendix tissue.
Positive control: Appendix (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 your samples.
⚠️Feasibility: No tissue is reported as not detected, so use siRNA knockdown or a KO line for a clean negative control.
HPA tissue expression evidence for CHAC1
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 → |
| Caudate | neuronal cells | High | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | neuronal cells | High | Protein (IHC) | HPA → |
| Epididymis | 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 | Low | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Low | Protein (IHC) | HPA → |
| Oral mucosa | squamous epithelial cells | Low | Protein (IHC) | HPA → |
| Skeletal muscle | myocytes | Low | Protein (IHC) | HPA → |
| Smooth muscle | smooth muscle cells | Low | Protein (IHC) | HPA → |
Section 3
Advanced CHAC1 Western Blot Tips
Deeper troubleshooting and optimisation questions for CHAC1, answered from its protein features.
How should CHAC1 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.
How could CHAC1 isoforms affect band interpretation?
Isoforms · Two isoforms are listed. Isoform 2 lacks residues 104–148 using the supplied UniProt coordinates, so it could migrate differently from isoform 1. Confirm which isoform the antibody recognizes before assigning either band.
Are annotated modifications likely to explain the higher band?
PTM · No modified residues or glycosylation sites are listed, and the glycosylation and disulfide counts are zero. These annotations provide no specific modification-based explanation for a 40 kDa band; they do not identify its cause.
Does the unfolded protein response suggest an induction experiment?
Induction · Unfolded protein response is a listed CHAC1 keyword. Compare a response-inducing condition with its control if testing expression changes, but the supplied features specify no inducer, dose, timing, or expected increase. Verify any change with a CHAC1-specific band.
What transfer method to use for CHAC1 Western blot?
Transfer · The predicted mass is 24.4 kDa, while the supplied observed band is approximately 40 kDa. The features do not prescribe a transfer method. Choose and check conditions that retain proteins across both sizes, using a molecular-weight marker and a post-transfer check.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A09017-2 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 CHAC1 bands be quantified across samples?
Quantitation · Quantify the same validated band across samples and keep the sampled fraction consistent. CHAC1 is listed in the cytosol and trans-Golgi network, so changes in fraction composition could affect the measured signal. Report separately any bands assigned to different isoforms.
Why might CHAC1 appear near 40 kDa instead of 24.4 kDa?
Interpretation · The supplied observed band is approximately 40 kDa, while the predicted mass is 24.4 kDa. The listed features do not establish the cause of that difference. Check whether the band is CHAC1 with an independent antibody or a CHAC1 depletion experiment before assigning it.
An interaction with NOTCH1 is listed, but that feature alone does not establish that an interaction survives Western blot preparation or creates a visible band. Test the band's identity directly before attributing an unexpected size to NOTCH1 binding.
An interaction with NOTCH1 is listed, but that feature alone does not establish that an interaction survives Western blot preparation or creates a visible band. Test the band's identity directly before attributing an unexpected size to NOTCH1 binding.
Boster reagents
CHAC1 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-CHAC1 Antibody Picoband®
Cat # A09017-2
A09017-2 is the listed anti-CHAC1 antibody, with reported human, mouse, and rat reactivity. Its WB image shows a band near 40 kDa in human, rat, and mouse cell lysates, although the expected CHAC1 size is 24 kDa; band identity remains uncertain.
Which to pick: A09017-2 is the only listed option. Its WB image uses human A549, HeLa, U251, and HEL; rat C6; and mouse RAW264.7 lysates at 1:1000 antibody dilution. Consider the 40 kDa versus 24 kDa size discrepancy when interpreting results.
Source: BosterBio CHAC1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9BUX1.
- Human Protein Atlas. CHAC1 tissue expression.
- PMC8311285 — target-verified WB comparison
- PMC10444662 — target-verified WB comparison
- PMC4908420 — target-verified WB comparison