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- Table of Contents
IK / Protein Red · Western blot design guide
Design a Western Blot for IK
Real validated IK Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-IK 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
IK 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 | ~65.6 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Adrenal gland (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 + Acetylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated IK Western Blot Protocols
The A03341 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | Target-positive lysate and matched negative control (standard starting point) |
| 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 | A03341; 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 IK Western Blot Band Size?
IK is predicted at 65.6 kDa; acetylation and phosphorylation could affect migration, but no empirical band size or modification-dependent shift is established.
| Band near 65.6 kDa in cell lysate | consistent with full-length IK; confirm identity with appropriate controls |
| Band near 65.6 kDa in a nuclear fraction | consistent with IK's nuclear localization |
| Band slightly above the main band | could reflect a modified IK state, but its migration is unverified |
| Close doublet near 65.6 kDa | could reflect different modification states; band identities require confirmation |
💡Expected IK appearanceIK has a predicted mass of 65.6 kDa; no empirical band size is supplied, and its listed modifications do not establish a visible migration shift, so confirm any candidate band with identity controls.
| UniProt predicted mass | places full-length IK near 65.6 kDa before any demonstrated migration effects |
| Lys98 acetylation | is documented, but its effect on apparent size is unknown |
| Lys137 acetylation | is documented, but its effect on apparent size is unknown |
| Ser287, Ser417, Ser460, Thr485 and Ser536 phosphorylation | may alter migration, but no shift is established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | IK may be poorly represented in the sampled fraction | check a nuclear fraction and verify extraction and antibody performance |
| Band higher than expected | a modification-related shift is possible but unproven | compare phosphatase-treated material and confirm identity by IK depletion |
| Band lower than expected | a smaller signal has no documented processing explanation here | check sample integrity and test whether the band decreases with IK depletion |
| Multiple bands | different modification states or antibody cross-reactivity are possible | compare phosphatase-treated material and use IK depletion to identify specific bands |
| Weak or no signal | the sampled fraction may contain little IK | compare whole-cell and nuclear fractions with suitable loading controls |
Experiment evidence
Western blot images showing IK band
A03341 · IK
A03341 · IK
A03341-1 · IK
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for IK Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for IK in Western blot, you can use adrenal gland lysate, supported by high HPA expression.
Positive control: Adrenal gland (IHC candidate; verify WB)
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside.
⚠️Feasibility: No tissue is listed as not detected, so use siRNA knockdown or a KO line for a negative control.
HPA tissue expression evidence for IK
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 |
|---|---|---|---|---|
| Adrenal gland | glandular cells | High | Protein (IHC) | HPA → |
| Appendix | glandular cells | High | Protein (IHC) | HPA → |
| Breast | glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | basal cells | High | Protein (IHC) | HPA → |
| Caudate | neuronal cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | adipocytes | Medium | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Cerebellum | cells in granular layer | Medium | Protein (IHC) | HPA → |
| Endometrium | glandular cells | Medium | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | Medium | Protein (IHC) | HPA → |
Section 3
Advanced IK Western Blot Tips
Deeper troubleshooting and optimisation questions for IK, answered from its protein features.
How should IK 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 IK isoforms account for multiple bands?
Isoforms · UniProt lists one IK isoform and no alternative sequence. Multiple bands should not be assigned to named isoforms on this evidence alone.
Which IK modifications are relevant when assessing band patterns?
PTM · The supplied UniProt coordinates list N6-acetyllysine at 98 and 137, phosphoserine at 287, 417, 460, and 536, and phosphothreonine at 485. These annotations identify possible modification states, but do not show that any causes a visible band shift. Keep the UniProt numbering explicit when comparing antibody or paper site labels.
Does influenza A infection imply IK induction?
Induction · IK is identified in a complex with SMU1 and influenza A virus polymerase subunits PB1 and PB2 during microbial infection. This supports considering infection status when comparing samples, but does not establish increased IK abundance or predict a band shift.
How should transfer be checked for IK?
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 A03341 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 IK Western blot signals be quantified?
Quantitation · Compare samples prepared from the same cellular fraction and normalize band signal to an appropriate loading measure. Because IK has several annotated locations, differences in fraction composition can affect signal without indicating a change in total cellular IK.
Where should IK migrate relative to its predicted mass?
Interpretation · IK has a predicted mass of 65.6 kDa. No observed band position is supplied, so use 65.6 kDa as a reference rather than an exact migration target. The listed modifications alone do not establish a visible shift or explain any difference from calculated mass.
IK is annotated in the nucleus, nucleoplasm, and chromosomes, and at cytoplasmic spindle poles. Compare like fractions when assessing band intensity; a difference between nuclear and cytoplasmic samples could reflect localization rather than a change in total IK abundance.
Check antibody specificity and sample conditions before assigning an unexpected band. UniProt lists no signal peptide, propeptide, glycosylation sites, or alternative sequence for IK. Its annotated modifications and infection associated complex do not, by themselves, identify the cause of an extra band.
IK is annotated in the nucleus, nucleoplasm, and chromosomes, and at cytoplasmic spindle poles. Compare like fractions when assessing band intensity; a difference between nuclear and cytoplasmic samples could reflect localization rather than a change in total IK abundance.
Check antibody specificity and sample conditions before assigning an unexpected band. UniProt lists no signal peptide, propeptide, glycosylation sites, or alternative sequence for IK. Its annotated modifications and infection associated complex do not, by themselves, identify the cause of an extra band.
Boster reagents
IK 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-Protein Red IK Antibody
Cat # A03341
Real WB data
Anti-IK/RED (P555) Antibody
Cat # A03341-1
The catalog reports two anti-IK antibodies, A03341 and A03341-1. Both report Human, Mouse, and Rat reactivity and have WB images. The supplied evidence covers the samples named in their captions; no publication or independent validation details are provided.
Which to pick: For rat brain, CT26, MCF-7, or H1792 samples, A03341-1 has a matching WB image at 1:500 with 40 µg per lane. For KB, SH-SY5Y, 293T, or 3T3, A03341 has a matching image. Both captions include HeLa.
Source: BosterBio IK gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q13123.
- Human Protein Atlas. IK tissue expression.
- PMC4177551 — target-verified WB comparison
- PMC8015194 — target-verified WB comparison
- PMC7198525 — target-verified WB comparison