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- Table of Contents
MITF / Microphthalmia-associated transcription factor · Western blot design guide
Design a Western Blot for MITF
Real validated MITF Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-MITF 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
MITF 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 | ~58.8 kDa | |
| Observed band | ~75 kDa | |
| Gel | 10% (catalog A00269-3) | |
| Positive control | Skin (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 | Phosphorylated + Ubl conjugation | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 12 isoform(s) |
Section 1
Real Curated MITF Western Blot Protocols
The A00269-3 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Jurkat, human Hela, human HEL (catalog A00269-3) |
| Gel % | 10% (catalog A00269-3) |
| Load | 30 ug; reducing conditions (catalog A00269-3) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A00269-3) |
| Membrane | nitrocellulose membrane (catalog A00269-3) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A00269-3) |
| Primary antibody | A00269-3 · 1:1000 (catalog A00269-3) |
| Primary incubation | overnight at 4°C (catalog A00269-3) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A00269-3) |
| Secondary incubation | 1.5 hour at RT (catalog A00269-3) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A00269-3) |
| Detection | ECL (catalog A00269-3) |
Section 2
What Is the Expected MITF Western Blot Band Size?
MITF is predicted at 58.8 kDa and observed near 75 kDa; the cause of the difference is not established.
| Band near 75 kDa | Empirical MITF band; its difference from the predicted mass is unexplained |
| Band near 59 kDa | Near the predicted 58.8 kDa mass; confirm its identity |
| Bands at different positions | Could reflect named MITF isoforms; their migration is not established |
| Doublet | Could reflect different phosphorylation states; test with phosphatase |
💡Expected MITF appearanceMITF has a predicted mass of 58.8 kDa, while an antibody QC blot reports a band near 75 kDa; the cause of the difference is unestablished, so confirm identity with appropriate controls.
| UniProt predicted mass | 58.8 kDa calculated; an empirical MITF band appears near 75 kDa |
| Isoforms A1, A2, B1, B2, C1, C2, H1, H2, M1, M2, Mdel, and 12 | May differ in size; distinct band positions are not established |
| Phosphoserine 180 by MAPK | May alter migration; a visible shift is not established |
| Phosphoserine 405 by GSK3 | May alter migration; a visible shift is not established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | MITF is found in the nucleus as well as other compartments | Check a nuclear fraction and verify antibody performance with a positive control |
| Band higher than expected | The observed 75 kDa band exceeds the 58.8 kDa prediction for an unestablished reason | Confirm band identity and compare phosphatase-treated samples |
| Band lower than expected | An alternative isoform is possible, but its mass is unknown | Check isoform recognition and confirm band identity |
| Multiple bands | MITF has multiple isoforms and phosphorylation sites, but distinct bands are unproven | Compare phosphatase-treated samples and validate bands with an independent antibody |
| Weak or no signal | MITF occurs in nuclear, cytoplasmic, and lysosome membrane compartments | Compare whole-cell and compartment-enriched samples with a positive control |
Experiment evidence
Western blot images showing MITF band
A00269-3 · MITF
M00269-2 · MITF
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for MITF Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for MITF in Western blot, you can use skin tissue, the highest-scoring HPA positive sample.
Positive control: Skin (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: Tissue controls are feasible because HPA reports high MITF in skin and no detection in adipose tissue.
HPA tissue expression evidence for MITF
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 |
|---|---|---|---|---|
| Skin | melanocytes | High | Protein (IHC) | HPA → |
| Appendix | germinal center cells | Medium | Protein (IHC) | HPA → |
| Colon | mucosal lymphoid cells | Medium | Protein (IHC) | HPA → |
| Endometrium | cells in endometrial stroma | Medium | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | 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 → |
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Breast | glandular 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 MITF Western Blot Tips
Deeper troubleshooting and optimisation questions for MITF, answered from its protein features.
What should be checked when unexpected MITF bands appear?
Band shift · Check whether their sizes and antibody epitopes are compatible with the annotated isoforms, especially the altered N termini and Mdel deletions. Also consider the listed phosphorylation sites, without assuming they cause a visible shift. MITF can occur in nuclear and cytoplasmic fractions, so compare like fractions before interpreting band differences.
How can MITF isoforms affect band interpretation?
Isoforms · UniProt lists 12 isoforms. M1, M2, and Mdel replace canonical residues 1–118; Mdel also lacks 139–194 and 293–298. Several other isoforms alter the N terminus or lack six residues near 294–299. Check which isoforms your antibody can recognize when interpreting bands of different sizes.
Yes. The canonical N terminus is replaced in the M, H, B, and C isoforms, while isoform 12 lacks residues 1–52. Compare the antibody epitope with the sequence of each isoform you intend to measure.
Yes. Mdel deletes canonical residues 139–194, which include the annotated MAPK site at 180. The M isoforms also replace canonical residues 1–118, which include the MTOR site at 5. Match phosphosite claims and antibody epitopes to the actual isoform sequence; coordinates here refer to canonical UniProt MITF.
Yes. The canonical N terminus is replaced in the M, H, B, and C isoforms, while isoform 12 lacks residues 1–52. Compare the antibody epitope with the sequence of each isoform you intend to measure.
Yes. Mdel deletes canonical residues 139–194, which include the annotated MAPK site at 180. The M isoforms also replace canonical residues 1–118, which include the MTOR site at 5. Match phosphosite claims and antibody epitopes to the actual isoform sequence; coordinates here refer to canonical UniProt MITF.
Which MITF phosphorylation sites matter when assessing bands?
PTM · Using canonical UniProt coordinates, phosphoserines are listed at 5 (MTOR), 180 (MAPK), 280 (MARK3), 405 (GSK3), 414, 491, and 516 (RPS6KA1). Antibody or paper numbering may differ. These annotations identify candidate sites to consider; they do not establish a visible shift or explain the observed mass.
Does this guide establish induction of MITF?
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 MITF?
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 A00269-3 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 MITF bands be quantified across samples?
Quantitation · Use the same defined band or set of bands across samples and state which you measured. MITF has 12 isoforms and is annotated in the nucleus, cytoplasm, and lysosome membrane, so keep the sampled cellular fraction consistent. A change in one band does not by itself establish a change in total MITF.
Why might MITF appear near 75 kDa instead of 58.8 kDa?
Interpretation · The supplied blot observation is approximately 75 kDa, while the predicted mass is 58.8 kDa. MITF has multiple isoforms and phosphorylation sites, but those features alone do not establish why this band migrates at 75 kDa. Compare the band with the expected isoform and antibody target before assigning it.
Boster reagents
MITF 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-MITF Antibody Picoband®
Cat # A00269-3
Real WB data
Anti-MiTF Rabbit Monoclonal Antibody
Cat # M00269-2
Two the supplier anti-MITF antibodies have WB images. A00269-3 shows human cell and rat and mouse heart lysates, with a band near 75 kDa versus an expected 59 kDa. M00269-2 shows an A375 lysate blot with limited caption detail.
Which to pick: Choose A00269-3 when its reported human cell or rat or mouse heart samples match your experiment; its caption provides conditions and band size. M00269-2 has an A375 lysate WB image, but its brief caption gives less detail for comparison.
Source: BosterBio MITF gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O75030.
- Human Protein Atlas. MITF tissue expression.
- PMC10918162 — target-verified WB comparison
- PMC6535666 — target-verified WB comparison
- PMC8221579 — target-verified WB comparison