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TRMT2A / tRNA (uracil-5-)-methyltransferase homolog A · Western blot design guide
Design a Western Blot for TRMT2A
Source-linked TRMT2A Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-TRMT2A WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Evidence assembled October 2026 · For research use; verify linked source records and product datasheet before use
TRMT2A 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 | ~68.7 kDa | |
| Observed band | 75 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Adipose tissue (IHC candidate; verify WB) +4 more | |
| Negative control | Liver (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Source-Linked TRMT2A Western Blot Protocol Options
The M13996-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | A431 cell lysate (catalog M13996-1) |
| 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 | M13996-1; 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 TRMT2A Western Blot Band Size?
TRMT2A is predicted at 68.7 kDa, while an empirical blot reports 75 kDa; the cause of that difference is not established.
| Band near 75 kDa | matches the reported empirical TRMT2A band; confirm identity with appropriate controls |
| Band near 68.7 kDa | near the predicted mass of full-length TRMT2A |
| Several bands | could reflect isoforms 1 and 2, but their migration is not established |
| Single band | does not rule out isoforms 1 and 2 because their separation is not established |
💡Expected TRMT2A appearanceTRMT2A has a predicted mass of 68.7 kDa and a reported empirical band at 75 kDa; the cause of the difference is unknown, so confirm band identity with appropriate controls.
| UniProt predicted mass | places full-length TRMT2A at 68.7 kDa by sequence calculation, versus the observed 75 kDa band |
| Isoform 1 | has no supplied isoform-specific mass or migration |
| Isoform 2 | has no supplied isoform-specific mass or migration |
| Alternative splicing | may alter protein size, but distinct band positions are not established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | cytosolic TRMT2A may be below detection or poorly recovered | check lysate quality and use a positive control |
| Band higher than expected | the reported 75 kDa band exceeds the 68.7 kDa prediction for an unknown reason | compare with a positive control and confirm identity by TRMT2A depletion |
| Band lower than expected | an alternative isoform is possible, but its migration is unknown | confirm identity by TRMT2A depletion and check antibody epitope coverage |
| Multiple bands | isoforms 1 and 2 are annotated, but distinct bands are unproven | compare with TRMT2A depletion and an independent antibody |
| Weak or no signal | low TRMT2A abundance or insufficient detection | check loading and antibody performance with a positive control |
Experiment evidence
Western blot images showing TRMT2A band
M13996-1 · TRMT2A
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for TRMT2A Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for TRMT2A in Western blot, you can use adipose tissue lysate, which shows high expression in HPA.
Positive control: Adipose tissue (IHC candidate; verify WB)
Negative control: Liver (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: Cytosolic TRMT2A can be assayed in tissue lysate, with HPA not detecting it in liver.
HPA tissue expression evidence for TRMT2A
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 |
|---|---|---|---|---|
| Adipose tissue | adipocytes | High | Protein (IHC) | HPA → |
| Appendix | glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | adipocytes | High | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Liver | cholangiocytes | Not detected | Protein (IHC) | HPA → |
| Caudate | glial cells | Low | Protein (IHC) | HPA → |
| Parathyroid gland | glandular cells | Low | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | endothelial cells | Medium | Protein (IHC) | HPA → |
Section 3
Advanced TRMT2A Western Blot Tips
Deeper troubleshooting and optimisation questions for TRMT2A, answered from its protein features.
How should TRMT2A 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 TRMT2A isoforms affect Western blot bands?
Isoforms · UniProt lists isoforms 1 and 2. In isoform 2, residues 550–562 are replaced and residues 563–625 are missing. An antibody recognizing the missing region may detect isoform 1 but miss isoform 2; an antibody recognizing a shared region could detect both. These sequence differences do not establish where either band will migrate.
Decide whether the measurement targets isoform 1 or both isoforms, then check whether the antibody recognizes sequence shared by both. Isoform 2 differs at residues 550–562 and lacks 563–625, so an antibody targeting that region could bias an isoform comparison. Quantify separately resolved bands separately rather than assuming they represent the same form.
Decide whether the measurement targets isoform 1 or both isoforms, then check whether the antibody recognizes sequence shared by both. Isoform 2 differs at residues 550–562 and lacks 563–625, so an antibody targeting that region could bias an isoform comparison. Quantify separately resolved bands separately rather than assuming they represent the same form.
Which TRMT2A phosphorylation sites matter when interpreting bands?
PTM · UniProt lists phosphoserine at positions 378 and 602 in its supplied sequence coordinates. Position 378 is retained in isoform 2, while position 602 falls within its missing 563–625 region. Check which isoform an antibody detects before interpreting a phosphorylation-related signal. The listed sites alone do not establish a visible band shift.
Does this guide establish induction of TRMT2A?
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 TRMT2A?
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 M13996-1 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 TRMT2A be quantified?
Quantitation · Standard workflow guidance: quantify only a validated target band within the linear exposure range. Use consistent sample preparation and loading, retain biological replicates, and avoid interpreting saturation or loading differences as regulation.
Why might TRMT2A appear at 75 kDa instead of 68.7 kDa?
Interpretation · The supplied apparent band is 75 kDa, versus a predicted mass of 68.7 kDa. TRMT2A has two listed phosphoserines and two isoforms, but these features alone do not establish the cause of the difference. Confirm band identity before assigning the 75 kDa signal to TRMT2A.
Yes. Isoform 2 replaces residues 550–562 and lacks residues 563–625. Check the antibody epitope against those regions: an epitope in the deleted region would exclude isoform 2, while one in the replaced region may recognize the isoforms differently. The supplied features cannot identify any particular unexpected band.
UniProt places TRMT2A in the cytoplasm, specifically the cytosol. A cytosolic sample is therefore relevant for detecting it. If a band appears in another fraction, the supplied location alone cannot establish what that band represents.
Yes. Isoform 2 replaces residues 550–562 and lacks residues 563–625. Check the antibody epitope against those regions: an epitope in the deleted region would exclude isoform 2, while one in the replaced region may recognize the isoforms differently. The supplied features cannot identify any particular unexpected band.
UniProt places TRMT2A in the cytoplasm, specifically the cytosol. A cytosolic sample is therefore relevant for detecting it. If a band appears in another fraction, the supplied location alone cannot establish what that band represents.
Boster reagents
TRMT2A 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-HTF9C Rabbit Monoclonal Antibody
Cat # M13996-1
The listed anti-TRMT2A antibody, M13996-1 (Anti-HTF9C Rabbit Monoclonal), is reported as reactive with human samples. Its Western blot image shows HTF9C expression in A431 cell lysate. No other sample contexts or publication evidence are supplied.
Which to pick: M13996-1 is the only listed option. It has a Western blot image using A431 cell lysate, making it the directly documented choice for that sample context; performance in other samples is not established here.
Source: BosterBio TRMT2A gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q8IZ69.
- Human Protein Atlas. TRMT2A tissue expression.
- PMC10484741 — target-verified WB comparison