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- Table of Contents
PUM2 / Pumilio homolog 2 · Western blot design guide
Design a Western Blot for PUM2
Source-linked PUM2 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-PUM2 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
PUM2 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 | ~114.2 kDa | |
| Observed band | ~114 kDa | |
| Gel | 10% (catalog M03347) | |
| 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 + Methylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 4 isoform(s) |
Section 1
Source-Linked PUM2 Western Blot Protocol Options
The M03347 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human 293T, human HEL, human SH-SY5Y, human REH, rat RH35, mouse HEPA1-6 (catalog M03347) |
| Gel % | 10% (catalog M03347) |
| Load | 30 ug; reducing conditions (catalog M03347) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog M03347) |
| Membrane | nitrocellulose membrane (catalog M03347) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog M03347) |
| Primary antibody | M03347 · 1:500 (catalog M03347) |
| Primary incubation | overnight at 4°C (catalog M03347) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog M03347) |
| Secondary incubation | 1.5 hour at RT (catalog M03347) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog M03347) |
| Detection | ECL (catalog M03347) |
Section 2
What Is the Expected PUM2 Western Blot Band Size?
PUM2 is predicted at 114.2 kDa and observed at ~114 kDa; its listed features do not establish a cause for the small difference.
| Band at ~114 kDa | matches the empirical PUM2 band and its 114.2 kDa predicted mass |
| Band near ~228 kDa | could reflect a PUM2 homodimer if it survives sample preparation |
| Several bands near ~114 kDa | could reflect PUM2 isoforms if their migration differs enough to resolve |
| Doublet near ~114 kDa | could reflect different phosphorylation states, pending validation |
💡Expected PUM2 appearancePUM2 has a predicted mass of 114.2 kDa and an empirical band at ~114 kDa in reducing whole-cell lysates; confirm identity with appropriate antibody controls.
| Predicted PUM2 mass | 114.2 kDa; the empirical band is ~114 kDa |
| PUM2 homodimerization in vitro | could yield a band near twice the monomer mass if the dimer survives preparation |
| Splice isoforms 1 and 2 | may differ in size, but their individual masses and migration are unspecified |
| Splice isoforms 3 and 4 | may differ in size, but their individual masses and migration are unspecified |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | PUM2 may be poorly recovered from cytoplasmic granules | check soluble and insoluble fractions and verify loading |
| Band higher than expected | a PUM2 homodimer may have survived sample preparation | compare denaturing preparations and confirm band identity |
| Band lower than expected | an isoform or PUM2 fragment may be detected | check antibody epitope coverage and use an independent antibody |
| Multiple bands | isoforms or different phosphorylation states may contribute | compare isoform expression and test phosphatase sensitivity |
| Weak or no signal | PUM2 abundance or extraction from cytoplasmic granules may be limiting | check loading and compare extraction fractions |
| Fragments below expected size | PUM2 may have degraded during preparation | repeat with protease inhibitors and compare freshly prepared lysate |
Experiment evidence
Western blot images showing PUM2 band
M03347 · PUM2
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for PUM2 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for PUM2 in Western blot, you can use appendix tissue, which HPA scores High.
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: Cytoplasmic localization supports tissue lysates; verify that HPA not-detected adipose tissue is negative on the blot.
HPA tissue expression evidence for PUM2
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 → |
| Colon | glandular cells | High | Protein (IHC) | HPA → |
| Esophagus | squamous epithelial cells | High | Protein (IHC) | HPA → |
| Gallbladder | glandular cells | High | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | 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 → |
| Bone marrow | hematopoietic cells | Low | Protein (IHC) | HPA → |
| Epididymis | glandular cells | Low | Protein (IHC) | HPA → |
| Oral mucosa | squamous epithelial cells | Low | Protein (IHC) | HPA → |
| Soft tissue | peripheral nerve | Low | Protein (IHC) | HPA → |
Section 3
Advanced PUM2 Western Blot Tips
Deeper troubleshooting and optimisation questions for PUM2, answered from its protein features.
How should PUM2 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 PUM2 isoforms produce additional bands?
Isoforms · Four isoforms are listed. Relative to the canonical sequence, isoform 2 lacks residues 574–652, isoform 4 lacks 1–56, and isoforms 3 and 4 lack 829–830. These sequence differences could affect migration; confirm which isoforms your antibody recognizes before assigning bands.
No. Isoform 2 lacks canonical residues 574–652, which include the listed phosphoserines at 587 and 592. Interpret site-specific signals against the isoform sequence; these positions use UniProt canonical numbering.
No. Isoform 2 lacks canonical residues 574–652, which include the listed phosphoserines at 587 and 592. Interpret site-specific signals against the isoform sequence; these positions use UniProt canonical numbering.
Which phosphorylation sites could affect PUM2 band interpretation?
PTM · UniProt lists phosphoserine at 67, 82, 102, 136, 178, 182, 587, 592, 684 and 700, plus phosphothreonine at 184 and 396. These are UniProt canonical coordinates; antibody or paper numbering may differ. Phosphatase treatment can test whether an observed band pattern depends on phosphorylation, but the listed sites do not establish a visible shift.
Does this guide establish induction of PUM2?
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 PUM2?
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 M03347 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 can PUM2 localization affect quantitation?
Quantitation · PUM2 is annotated in the cytoplasm, perinuclear region and cytoplasmic stress granules. Compare samples prepared with the same fractionation and extraction procedure, since differing recovery from these compartments could change the measured signal.
Should PUM2 migrate above its predicted mass?
Interpretation · The supplied Western blot band is approximately 114 kDa, close to the canonical predicted mass of 114.2 kDa. The listed modifications alone do not establish a visible shift or explain any mass difference.
Check the antibody epitope against the listed deletions: residues 1–56 are absent from isoform 4, 574–652 from isoform 2, and 829–830 from isoforms 3 and 4. An epitope within a deleted region cannot detect that isoform.
Yes. UniProt lists omega-N-methylarginine at canonical Arg674. This annotation supports checking the site when interpreting a methylation-specific result, but does not establish a detectable Western blot shift. Confirm numbering conventions when comparing antibodies or papers.
Compare bands with the approximately 114 kDa observed band and the 114.2 kDa canonical prediction. Check whether the antibody epitope is present in each of the four isoforms, then assess whether phosphorylation contributes to the pattern. The supplied features alone cannot identify an unexpected band.
Check the antibody epitope against the listed deletions: residues 1–56 are absent from isoform 4, 574–652 from isoform 2, and 829–830 from isoforms 3 and 4. An epitope within a deleted region cannot detect that isoform.
Yes. UniProt lists omega-N-methylarginine at canonical Arg674. This annotation supports checking the site when interpreting a methylation-specific result, but does not establish a detectable Western blot shift. Confirm numbering conventions when comparing antibodies or papers.
Compare bands with the approximately 114 kDa observed band and the 114.2 kDa canonical prediction. Check whether the antibody epitope is present in each of the four isoforms, then assess whether phosphorylation contributes to the pattern. The supplied features alone cannot identify an unexpected band.
Boster reagents
PUM2 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-Pumilio 2 Rabbit Monoclonal Antibody
Cat # M03347
M03347 is a rabbit monoclonal anti-PUM2 antibody listed for human, mouse, and rat. Its Western blot image reports a band near the expected 114 kDa in the specified cell lysates; no publication evidence was supplied.
Which to pick: M03347 is the only listed option and has a Western blot image. Its caption documents human 293T, HEL, SH-SY5Y, and REH; rat RH35; and mouse HEPA1-6 whole cell lysates, using 30 µg per lane and a 1:500 primary dilution.
Source: BosterBio PUM2 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q8TB72.
- Human Protein Atlas. PUM2 tissue expression.
- PMC9434726 — target-verified WB comparison
- PMC6114944 — target-verified WB comparison
- PMC10865267 — target-verified WB comparison