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- Table of Contents
SHMT2 / Serine hydroxymethyltransferase, mitochondrial · Western blot design guide
Design a Western Blot for SHMT2
Real validated SHMT2 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-SHMT2 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
SHMT2 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 | ~56 kDa | |
| Observed band | ~56 kDa | |
| Gel | 10% (catalog A03233-1) | |
| Positive control | Appendix (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 + Acetylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 3 isoform(s) |
Section 1
Real Curated SHMT2 Western Blot Protocols
The A03233-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human HEL, human K562, human HepG2 (catalog A03233-1) |
| Gel % | 10% (catalog A03233-1) |
| Load | 30 ug; reducing conditions (catalog A03233-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A03233-1) |
| Membrane | nitrocellulose membrane (catalog A03233-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A03233-1) |
| Primary antibody | A03233-1 · 1:1000 (catalog A03233-1) |
| Primary incubation | overnight at 4°C (catalog A03233-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A03233-1) |
| Secondary incubation | 1.5 hour at RT (catalog A03233-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A03233-1) |
| Detection | ECL (catalog A03233-1) |
Section 2
What Is the Expected SHMT2 Western Blot Band Size?
SHMT2 is predicted at 56 kDa and observed near 56 kDa; isoforms and oligomers are documented, but distinct migration effects are unestablished.
| Band near 56 kDa | Matches the predicted mass and the observed SHMT2 band in reducing whole-cell blots. |
| Higher band | Could reflect an SHMT2 oligomer only if it survives sample denaturation. |
| Several bands at different sizes | Could reflect SHMT2 isoforms 1, 2, and 3; their migration differences are unknown. |
| Weak or absent band in a mitochondria-depleted fraction | Consistent with SHMT2's predominantly mitochondrial localization. |
💡Expected SHMT2 appearanceSHMT2 has a predicted mass of 56 kDa and an empirical band near 56 kDa in reducing whole-cell blots; confirm band identity with appropriate antibody controls.
| Predicted SHMT2 mass | Places the full-length sequence near 56 kDa. |
| Isoforms 1, 2, and 3 | May differ in size, but their individual masses and migration are not supplied. |
| PLP-bound homotetramer | Could produce a higher apparent band only if the complex survives sample preparation. |
| PLP-free homodimer | Could produce a higher apparent band only if the complex survives sample preparation. |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | SHMT2 signal may be low in the sampled material or lost during preparation. | Check loading and transfer, and compare with a positive whole-cell lysate. |
| Band higher than expected | A persistent SHMT2 oligomer is possible, but its migration is unverified. | Check denaturation and reduction, then verify identity with an independent antibody. |
| Band lower than expected | An alternative isoform is possible, but no isoform mass is supplied. | Check band identity with an independent antibody and compare samples expressing SHMT2. |
| Multiple bands | SHMT2 has three named isoforms, though distinct bands are not established. | Compare bands across samples and confirm them with an independent antibody. |
| Weak or no signal | The sampled fraction may contain little mitochondria-associated SHMT2. | Compare with whole-cell or mitochondria-enriched lysate and check loading. |
Experiment evidence
Western blot images showing SHMT2 band
A03233-1 · SHMT2
A03233-1 · SHMT2
A03233-1 · SHMT2
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for SHMT2 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for SHMT2 in Western blot, you can use appendix tissue, which HPA rates high for SHMT2.
Positive control: Appendix (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: HPA identifies both a high-expression tissue and a not-detected tissue, making tissue controls feasible.
HPA tissue expression evidence for SHMT2
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 | lymphoid tissue | High | Protein (IHC) | HPA → |
| Bronchus | ciliated cells (cell body) | High | Protein (IHC) | HPA → |
| Colon | glandular cells | High | Protein (IHC) | HPA → |
| Kidney | proximal tubules (cell body) | High | Protein (IHC) | HPA → |
| Liver | hepatocytes | 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 → |
| Heart muscle | cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Ovary | ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
| Skeletal muscle | myocytes | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | smooth muscle cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced SHMT2 Western Blot Tips
Deeper troubleshooting and optimisation questions for SHMT2, answered from its protein features.
How should SHMT2 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 SHMT2 isoforms produce different bands?
Isoforms · UniProt lists three isoforms. Isoform 2 lacks residues 199–208, while isoform 3 lacks residues 1–21, using canonical UniProt numbering. These sequence differences could affect apparent mass, but the supplied features do not establish whether the isoforms resolve as separate bands.
Which SHMT2 modifications matter when examining band differences?
PTM · UniProt lists acetyllysines at 103, 181, 196, 297, 356, 464, 469 and 474, plus phosphoserine at 470. These are canonical UniProt coordinates, which may differ from paper or antibody numbering. Their presence alone does not establish a visible band shift.
Canonical UniProt Lys280 has alternate annotations for pyridoxal phosphate binding and succinylation. Keep those assignments distinct when interpreting an antibody targeting this region; the annotations do not show that either state produces a separate Western blot band.
Canonical UniProt Lys280 has alternate annotations for pyridoxal phosphate binding and succinylation. Keep those assignments distinct when interpreting an antibody targeting this region; the annotations do not show that either state produces a separate Western blot band.
Does this guide establish induction of SHMT2?
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 SHMT2?
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 A03233-1 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 SHMT2 bands be quantified across samples?
Quantitation · Quantify the same defined band region across samples and report whether it includes any resolved isoform bands. SHMT2 has three annotated isoforms, so a single band measurement may not distinguish them. Keep the band selection consistent when comparing samples.
How should the ~56 kDa band be interpreted?
Interpretation · SHMT2 has a predicted mass of 56 kDa, matching the supplied observed band of approximately 56 kDa. Mass agreement supports the assignment but does not establish band identity; compare with an appropriate SHMT2 control.
UniProt places SHMT2 mainly in mitochondria and also lists cytoplasm and nucleus. Compare the band across appropriately validated fractions when location is relevant. Location alone cannot establish that an unexpected band is SHMT2.
UniProt describes SHMT2 as a homotetramer with bound pyridoxal phosphate and a homodimer without it. Those assembly states do not, by themselves, identify additional Western blot bands. Check whether a band tracks with SHMT2 in a suitable control before assigning it to a complex or isoform.
UniProt places SHMT2 mainly in mitochondria and also lists cytoplasm and nucleus. Compare the band across appropriately validated fractions when location is relevant. Location alone cannot establish that an unexpected band is SHMT2.
UniProt describes SHMT2 as a homotetramer with bound pyridoxal phosphate and a homodimer without it. Those assembly states do not, by themselves, identify additional Western blot bands. Check whether a band tracks with SHMT2 in a suitable control before assigning it to a complex or isoform.
Boster reagents
SHMT2 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-SHMT2 Antibody Picoband®
Cat # A03233-1
The catalog reports one anti-SHMT2 antibody, A03233-1. Its Western blot image reports an approximately 56 kDa band in named human, rat, and mouse cell lysates. The supplied evidence is limited to this product image and its stated conditions.
Which to pick: A03233-1 is the only listed option. Its catalogued reactivity is human, mouse, and rat. The WB caption documents HEL, K562, HepG2, Jurkat, C6, RAW264.7, and ANA-1 lysates using a 1:1000 antibody dilution under reducing conditions.
Source: BosterBio SHMT2 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P34897.
- Human Protein Atlas. SHMT2 tissue expression.
- PMC7648565 — target-verified WB comparison
- PMC7806468 — target-verified WB comparison
- PMC11914208 — target-verified WB comparison