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- Table of Contents
MLH3 / DNA mismatch repair protein Mlh3 · Western blot design guide
Design a Western Blot for MLH3
Source-linked MLH3 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-MLH3 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
MLH3 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 | ~163.7 kDa | |
| Gel | 8–10% (standard starting point) | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | — | |
| Caveat | Peptide-blocking control | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Source-Linked MLH3 Western Blot Protocol Options
The A04640 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | lysates from K562 and HT-29 cells, (catalog A04640) |
| Gel % | 8–10% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Wet/tank transfer; optimize duration (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | the synthesized peptide (catalog A04640) |
| Primary antibody | A04640; 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 MLH3 Western Blot Band Size?
MLH3 is predicted at 163.7 kDa; isoforms could affect migration, but no empirical band size or distinct isoform pattern is supplied.
| Band near 163.7 kDa | Consistent with the predicted MLH3 mass; confirm identity with controls |
| Single band near 163.7 kDa | Compatible with MLH3 even though two isoforms are listed |
| Multiple bands | Isoforms 1 and 2 are possible contributors, but distinct migration is unverified |
| Band enriched in nuclear extract | Consistent with MLH3's nuclear location |
💡Expected MLH3 appearanceUniProt predicts MLH3 at 163.7 kDa, but no empirical band size is supplied; use band-identity controls because the migration of isoforms 1 and 2 is unknown.
| UniProt predicted mass | Places the reference size at 163.7 kDa, without establishing apparent migration |
| Isoform 1 | Its individual mass and migration are not supplied |
| Isoform 2 | Its individual mass and migration are not supplied |
| Alternative splicing | Could alter isoform size, but a resolvable difference is not established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Nuclear MLH3 may be poorly recovered in the extract | Check nuclear extraction and a nuclear protein control |
| Band higher than expected | No supplied feature establishes a higher MLH3 band | Compare with a molecular-weight marker and verify antibody specificity |
| Band lower than expected | An isoform or unrelated signal is possible; isoform masses are unknown | Check antibody specificity and compare independent MLH3 antibodies |
| Multiple bands | Two isoforms are listed, but their migration is unknown | Compare isoform-specific evidence and use a peptide-blocking control |
| Weak or no signal | Nuclear MLH3 may be underrepresented in the sample | Test a nuclear-enriched extract and confirm protein loading |
Experiment evidence
Western blot images showing MLH3 band
A04640 · MLH3
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for MLH3 Western blot
🧪For positive controls for MLH3 in Western blot, you can use a validated MLH3-expressing sample; the supplied HPA evidence identifies no positive tissue or cell line.
Positive control: No high/medium HPA tissue identified
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: MLH3 is nuclear, but without HPA expression data, suitable positive and negative samples need validation.
HPA tissue expression evidence for MLH3
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 |
|---|---|---|---|---|
| No high/medium HPA tissues identified in the supplied evidence. | ||||
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| No lower-expression tissue rows available in the supplied evidence. | ||||
Section 3
Advanced MLH3 Western Blot Tips
Deeper troubleshooting and optimisation questions for MLH3, answered from its protein features.
Where should the MLH3 band appear?
Band shift · The predicted mass is 163.7 kDa. No observed Western blot band is supplied, so use this as a reference rather than an established apparent mass.
Could MLH3 isoforms produce different bands?
Isoforms · UniProt lists two isoforms. Isoform 2 lacks canonical residues 1215–1238, so it may migrate differently. Those coordinates use the supplied canonical sequence; a visible separation is not established.
Do annotated modifications prove a band shift?
PTM · The linked UniProt record describes protein features. A modification annotation alone does not demonstrate a visible shift; retain any condition or experimental qualifier attached to it.
Does this guide establish induction of MLH3?
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 MLH3 Western blot?
Transfer · MLH3 is predicted to be 163.7 kDa. Check transfer efficiency at that size with a suitable high-mass control and optimize conditions empirically. The supplied features specify no transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A04640 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 MLH3 signals be quantified?
Quantitation · Measure the same MLH3 band consistently across samples and normalize to an appropriate loading measure for the fraction analyzed. If two isoform bands resolve, quantify them separately.
Which sample fraction is relevant for MLH3?
Interpretation · MLH3 is annotated as nuclear. A nuclear fraction may help assess its signal; compare equivalent fractions when interpreting differences between samples.
Consider the two annotated isoforms, including isoform 2's deletion of canonical residues 1215–1238. The supplied features do not establish where either isoform migrates or assign other bands to a modification.
MLH3 forms a heterodimer with MLH1, but that interaction alone does not establish the identity of an extra Western blot band. Confirm band identity independently.
Consider the two annotated isoforms, including isoform 2's deletion of canonical residues 1215–1238. The supplied features do not establish where either isoform migrates or assign other bands to a modification.
MLH3 forms a heterodimer with MLH1, but that interaction alone does not establish the identity of an extra Western blot band. Confirm band identity independently.
Boster reagents
MLH3 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-MLH3 Antibody
Cat # A04640
The catalog reports one anti-MLH3 antibody for Western blotting, A04640, with reported human reactivity. Its WB image shows K562 and HT-29 cell lysates and a lane blocked with the synthesized peptide. Evidence here is limited to that product record and image caption.
Which to pick: A04640 is the only listed option. Choose it for a human MLH3 Western blot if its reported K562 and HT-29 lysate context fits your experiment; it has a WB image with a peptide-blocked lane.
Source: BosterBio MLH3 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9UHC1.
- Human Protein Atlas. MLH3 tissue expression.
- PMC5522225 — target-verified WB comparison