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- Table of Contents
ALK / ALK tyrosine kinase receptor · Western blot design guide
Design a Western Blot for ALK
Real validated ALK Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-ALK 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
ALK 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 | ~176.4 kDa | |
| Observed band | ~250 kDa | |
| Gel | 8% (catalog PA1741) | |
| Positive control | Cerebellum (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 | Glycosylated + Phosphorylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated ALK Western Blot Protocols
The PA1741 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human SH-SY5Y (catalog PA1741) |
| Gel % | 8% (catalog PA1741) |
| Load | 30 ug; reducing conditions (catalog PA1741) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog PA1741) |
| Membrane | nitrocellulose membrane (catalog PA1741) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog PA1741) |
| Primary antibody | PA1741 · 0.5 μg/mL (catalog PA1741) |
| Primary incubation | overnight at 4°C (catalog PA1741) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog PA1741) |
| Secondary incubation | 1.5 hour at RT (catalog PA1741) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog PA1741) |
| Detection | ECL (catalog PA1741) |
Section 2
What Is the Expected ALK Western Blot Band Size?
ALK is predicted at 176.4 kDa, while a reducing whole-cell blot shows ~250 kDa; the cause of the difference is not established.
| Band near 250 kDa | Matches the empirical ALK band in reducing whole-cell lysate; confirm identity with controls |
| Band near 176.4 kDa | Near the predicted full-length sequence mass; migration at this position is unverified |
| Band slightly below the precursor | Could reflect removal of the 18-residue signal peptide |
| Higher band under non-reducing conditions | Could reflect an ALK homodimer if it survives electrophoresis |
| Diffuse band | Could reflect heterogeneous N-linked glycosylation; site annotations alone do not establish a smear |
💡Expected ALK appearanceALK has a predicted sequence mass of 176.4 kDa, while a reducing whole-cell blot shows a band near 250 kDa; confirm band identity with ordinary controls because the cause of this difference is unproven.
| Predicted sequence mass | 176.4 kDa is the full-length sequence baseline, not a validated migration position |
| N-linked glycosylation at Asn169 and other annotated sites | Could increase apparent mass, but the observed shift is not established as glycosylation |
| Signal peptide at residues 1–18 | Cleavage makes the mature chain slightly smaller than the precursor |
| Ligand-induced ALK homodimer | Could produce a higher band if the dimer survives sample preparation and electrophoresis |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | The ~250 kDa empirical band exceeds the 176.4 kDa sequence prediction; its cause is unproven | Compare with the reported ~250 kDa band and verify identity using ALK knockdown or a second antibody |
| Band lower than expected | Signal-peptide removal may slightly reduce mass; a larger decrease is unexplained | Check band identity with ALK knockdown or a second antibody |
| Broad smear instead of sharp band | Heterogeneous N-linked glycosylation is possible | Compare treated and untreated samples after N-glycan removal, alongside an ALK identity control |
| Multiple bands | Different glycosylation or processing states are possible but no distinct masses are supplied | Check which bands respond to ALK knockdown and compare reducing conditions |
| Weak or no signal | ALK is a single-pass membrane protein and may be poorly recovered from lysate | Check membrane-protein extraction and use a positive lysate control |
Experiment evidence
Western blot images showing ALK band
PA1741 · ALK
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for ALK Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for ALK in Western blot, you can use cerebellum tissue, which HPA scores high for ALK.
Positive control: Cerebellum (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: ALK is a membrane protein, so membrane-enriched lysate may improve detection.
HPA tissue expression evidence for ALK
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 |
|---|---|---|---|---|
| Cerebellum | Purkinje cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | neuronal cells | High | Protein (IHC) | HPA → |
| Hippocampus | neuronal cells | High | Protein (IHC) | HPA → |
| Caudate | neuronal cells | Medium | Protein (IHC) | HPA → |
| Skin | keratinocytes | Medium | 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 → |
| Appendix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
| Duodenum | glandular cells | Not detected | Protein (IHC) | HPA → |
| Esophagus | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ALK Western Blot Tips
Deeper troubleshooting and optimisation questions for ALK, answered from its protein features.
How should ALK 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 ALK isoforms explain additional bands?
Isoforms · The supplied record lists one isoform and no alternative sequence. It provides no basis for assigning an additional band to a specific ALK isoform.
Which ALK phosphorylation sites can guide phospho-specific detection?
PTM · The annotated phosphotyrosines are at UniProt positions 1078, 1092, 1096, 1131, 1278, 1507, and 1604. Match the antibody’s stated site to UniProt numbering; antibody or paper numbering may differ. These annotations do not establish that phosphorylation will produce a visible band shift.
ALK has seven annotated phosphotyrosines. Compare blocking conditions using the intended phospho-specific antibody and matched samples, choosing the condition that gives a clear ALK signal with low background. The supplied features do not identify a preferred blocker.
Measure the phospho-specific signal alongside total ALK in matched samples and account for loading. ALK has multiple annotated phosphotyrosines, so identify the site recognized by the antibody. Use the same band region across samples; the approximately 250 kDa observation does not establish that every ALK species migrates there.
ALK has seven annotated phosphotyrosines. Compare blocking conditions using the intended phospho-specific antibody and matched samples, choosing the condition that gives a clear ALK signal with low background. The supplied features do not identify a preferred blocker.
Measure the phospho-specific signal alongside total ALK in matched samples and account for loading. ALK has multiple annotated phosphotyrosines, so identify the site recognized by the antibody. Use the same band region across samples; the approximately 250 kDa observation does not establish that every ALK species migrates there.
What stimulation condition is supported for an ALK comparison?
Induction · ALK is annotated to homodimerize following heparin and ligand binding. A matched comparison with and without heparin and ligand can examine ALK or site-specific phosphotyrosine signal. The supplied features do not establish the size or direction of a phosphorylation change under those conditions.
What transfer method to use for ALK Western blot?
Transfer · The observed ALK band is approximately 250 kDa, and ALK is a single-pass membrane protein. Check transfer at the high-mass marker and assess signal remaining in the gel when optimizing transfer. The supplied features do not specify a transfer method or settings.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the PA1741 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 ALK 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 ALK appear near 250 kDa instead of 176.4 kDa?
Interpretation · The observed band is approximately 250 kDa, while the predicted sequence mass is 176.4 kDa. ALK has 16 annotated N-linked glycosylation sites and a signal peptide at residues 1–18. These features motivate checking glycosylation, but their presence alone does not establish the cause or size of the apparent mass difference.
Check whether bands are detected by both total and site-specific ALK antibodies. The record lists 16 N-linked glycosylation sites, seven phosphotyrosines, and a chromosomal rearrangement keyword, but supplies no sizes for alternative products. None of these annotations alone identifies an unexpected band or proves a visible shift.
Check whether bands are detected by both total and site-specific ALK antibodies. The record lists 16 N-linked glycosylation sites, seven phosphotyrosines, and a chromosomal rearrangement keyword, but supplies no sizes for alternative products. None of these annotations alone identifies an unexpected band or proves a visible shift.
Boster reagents
ALK 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-ALK Antibody Picoband®
Cat # PA1741
PA1741 is an anti-ALK antibody listed for human, mouse, and rat. Its WB image uses human SH-SY5Y whole-cell lysate and shows a band near 250 kDa, versus an expected 176 kDa; the band discrepancy limits interpretation.
Which to pick: PA1741 is the only listed option. Its WB image provides a human SH-SY5Y example at 0.5 μg/mL. Mouse and rat reactivity is listed, but no WB examples for those species are supplied.
Source: BosterBio ALK gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9UM73.
- Human Protein Atlas. ALK tissue expression.
- PMC13130619 — target-verified WB comparison
- PMC4529334 — target-verified WB comparison
- PMC8833940 — target-verified WB comparison