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- Table of Contents
HIPK2 / Homeodomain-interacting protein kinase 2 · Western blot design guide
Design a Western Blot for HIPK2
Real validated HIPK2 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-HIPK2 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
HIPK2 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 | ~131 kDa | |
| Observed band | ~100 kDa | |
| Gel | 5–20% (catalog M01371) | |
| Positive control | Adipose tissue (IHC candidate; verify WB) +4 more | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Ubl conjugation | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 3 isoform(s) |
Section 1
Real Curated HIPK2 Western Blot Protocols
The M01371 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human HT-1080, human Raji, human PC-3 (catalog M01371) |
| Gel % | 5–20% (catalog M01371) |
| Load | 30 ug; reducing conditions (catalog M01371) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog M01371) |
| Membrane | nitrocellulose membrane (catalog M01371) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog M01371) |
| Primary antibody | M01371 · 1:500 (catalog M01371) |
| Primary incubation | overnight at 4°C (catalog M01371) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:500 (catalog M01371) |
| Secondary incubation | 1.5 hour at RT (catalog M01371) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog M01371) |
| Detection | ECL (catalog M01371) |
Section 2
What Is the Expected HIPK2 Western Blot Band Size?
HIPK2 is predicted at 131 kDa, while a band near 100 kDa was observed in reducing whole-cell lysates; the cause of the difference is not established.
| Band near 100 kDa | Empirical HIPK2 signal reported in reducing whole-cell lysates; confirm identity with controls |
| Band near 131 kDa | Candidate near the predicted HIPK2 mass; confirm identity with controls |
| Several bands at different positions | Isoforms 1, 2 and 3 are annotated, but their migration is not established |
| Closely spaced bands | Phosphorylated HIPK2 states are possible, but a visible shift is not established |
💡Expected HIPK2 appearanceHIPK2 has a predicted mass of 131 kDa and an empirical band near 100 kDa in reducing whole-cell lysates; the difference is unexplained, so confirm band identity with an appropriate positive control and HIPK2 depletion.
| Predicted HIPK2 mass | 131 kDa is the sequence-based reference, while the reported band is near 100 kDa |
| Isoform 1 | Its apparent size relative to the other isoforms is not supplied |
| Isoform 2 | Alternative splicing could change size, but its relative size is not supplied |
| Isoform 3 | Alternative splicing could change size, but its relative size is not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | HIPK2 is concentrated in nuclear bodies, so whole-cell recovery may be limiting | Check a nuclear fraction and a positive control lysate |
| Band higher than expected | An isoform or phosphorylated state is possible, but neither explains a specific position from the supplied evidence | Compare with a positive control and confirm identity by HIPK2 depletion |
| Band lower than expected | The reported 100 kDa band is below the 131 kDa prediction for an undetermined reason | Compare with the reported positive control lysates and confirm identity by HIPK2 depletion |
| Multiple bands | Annotated isoforms or phosphorylation states could contribute, but distinct migration is unverified | Check which bands respond to HIPK2 depletion; consider phosphatase treatment for close bands |
| Weak or no signal | Whole-cell lysate may yield less HIPK2 signal than a nuclear fraction | Check nuclear enrichment and include a positive control lysate |
Experiment evidence
Western blot images showing HIPK2 band
M01371 · HIPK2
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for HIPK2 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for HIPK2 in Western blot, you can use adipose tissue lysate, which has high HPA expression.
Positive control: Adipose tissue (IHC candidate; verify WB)
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: No tissue is reported as not detected, so use siRNA knockdown or a KO line for a negative control.
HPA tissue expression evidence for HIPK2
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 | lymphoid tissue | High | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Caudate | glial cells | High | Protein (IHC) | HPA → |
| Cerebellum | cells in molecular layer | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | glandular cells | Medium | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Breast | adipocytes | Medium | Protein (IHC) | HPA → |
| Endometrium | glandular cells | Medium | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | Medium | Protein (IHC) | HPA → |
Section 3
Advanced HIPK2 Western Blot Tips
Deeper troubleshooting and optimisation questions for HIPK2, answered from its protein features.
How should HIPK2 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 HIPK2 isoforms affect band interpretation?
Isoforms · UniProt lists three isoforms. Relative to the canonical sequence, isoform 3 lacks residues 595–621. Isoform 2 lacks 808–907 and 1019–1198, and replaces 989–1018 with a different sequence. Check whether the antibody recognizes a region retained in the isoform being measured. Coordinates here use the supplied canonical UniProt sequence.
Check the antibody epitope against the supplied canonical UniProt coordinates. Isoform 3 lacks 595–621; isoform 2 lacks 808–907 and 1019–1198 and replaces 989–1018. An epitope in one of those regions may prevent detection of that isoform. Account for the altered sequence when comparing antibody results.
Check the antibody epitope against the supplied canonical UniProt coordinates. Isoform 3 lacks 595–621; isoform 2 lacks 808–907 and 1019–1198 and replaces 989–1018. An epitope in one of those regions may prevent detection of that isoform. Account for the altered sequence when comparing antibody results.
Could phosphorylation shift the HIPK2 band?
PTM · UniProt lists 21 modified residues, including phosphotyrosine at canonical Tyr361 by autocatalysis. Phosphorylation may affect migration, but the listed sites do not demonstrate a visible shift or account for the approximately 100 kDa band. Compare matched samples with and without phosphatase treatment if testing a phosphorylation-dependent mobility change.
Does this guide establish induction of HIPK2?
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 HIPK2 Western blot?
Transfer · The canonical HIPK2 protein is predicted at 131 kDa, and the reported apparent band is approximately 100 kDa. Use a transfer setup that captures proteins across that range, then check transfer at both positions. The supplied features do not establish a specific membrane, buffer, or transfer duration.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the M01371 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 HIPK2 bands be quantified?
Quantitation · Define the band or bands being measured before comparing samples. Three isoforms and 21 listed phosphorylation sites complicate assigning every signal to the same HIPK2 species. Use the same band boundary across lanes and verify that the antibody recognizes the relevant isoform; report separate bands separately when their identities remain uncertain.
Why might HIPK2 appear near 100 kDa instead of 131 kDa?
Interpretation · The canonical protein has a predicted mass of 131 kDa, while the reported band is approximately 100 kDa. Isoform 2 lacks substantial sequence, making it a candidate explanation. Confirm the isoform and antibody target before assigning the band; the listed features alone do not establish its identity or explain the full mass difference.
First compare their positions with the approximately 100 kDa reported band and the 131 kDa canonical prediction. Check whether the antibody epitope is retained in isoforms 2 and 3. HIPK2 is concentrated in nuclear PML bodies, with small cytoplasmic amounts, so compare the sample fraction used. These features provide possibilities, not band identities.
First compare their positions with the approximately 100 kDa reported band and the 131 kDa canonical prediction. Check whether the antibody epitope is retained in isoforms 2 and 3. HIPK2 is concentrated in nuclear PML bodies, with small cytoplasmic amounts, so compare the sample fraction used. These features provide possibilities, not band identities.
Boster reagents
HIPK2 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-HIPK2 Rabbit Monoclonal Antibody
Cat # M01371
The catalog reports one anti-HIPK2 antibody, rabbit monoclonal M01371, with reported human and mouse reactivity. Its WB image shows human HT-1080, Raji, PC-3, and HEL lysates. The observed band is approximately 100 kDa, while the expected size is 130 kDa.
Which to pick: M01371 is the only listed option and has a WB image from human cell lysates tested at 1:500. Mouse reactivity is reported, but no mouse WB example is supplied. Account for the observed 100 kDa band versus the expected 130 kDa size.
Source: BosterBio HIPK2 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9H2X6.
- Human Protein Atlas. HIPK2 tissue expression.
- PMC3117790 — target-verified WB comparison
- PMC10503042 — target-verified WB comparison
- PMC9510922 — target-verified WB comparison