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NCK1 / SH2/SH3 adapter protein NCK1 · Western blot design guide
Design a Western Blot for NCK1
Source-linked NCK1 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-NCK1 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
NCK1 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 | ~42.9 kDa | |
| Observed band | ~43 kDa | |
| Gel | 10% (catalog M02260) | |
| Positive control | Adrenal gland (IHC candidate; verify WB) +4 more | |
| Negative control | Caudate (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Acetylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Source-Linked NCK1 Western Blot Protocol Options
The M02260 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Hela, human SIHA, human A431, human Jurkat, rat brain, rat liver, mouse brain, mouse liver (catalog M02260) |
| Gel % | 10% (catalog M02260) |
| Load | 30 ug; reducing conditions (catalog M02260) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog M02260) |
| Membrane | nitrocellulose membrane (catalog M02260) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog M02260) |
| Primary antibody | M02260 · 1:500 (catalog M02260) |
| Primary incubation | overnight at 4°C (catalog M02260) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog M02260) |
| Secondary incubation | 1.5 hour at RT (catalog M02260) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog M02260) |
| Detection | ECL (catalog M02260) |
Section 2
What Is the Expected NCK1 Western Blot Band Size?
NCK1 is predicted at 42.9 kDa and observed at ~43 kDa; the small difference has no established cause.
| Band at ~43 kDa | Matches the empirical NCK1 band and its 42.9 kDa predicted mass |
| Additional band near ~43 kDa | Could reflect isoform 1 or 2, but their migration is not established |
| Slightly shifted band | Could reflect NCK1 phosphorylation; a visible shift is not established |
| Weak cytoplasmic band after genotoxic stress | May reflect NCK1 redistribution to the nucleus |
💡Expected NCK1 appearanceNCK1 is predicted at 42.9 kDa and observed at ~43 kDa; confirm band identity with an appropriate antibody control or NCK1 depletion.
| Predicted NCK1 mass | 42.9 kDa, consistent with the observed ~43 kDa band |
| Isoform 1 | Its mass and migration relative to isoform 2 are not supplied |
| Isoform 2 | Its mass and migration relative to isoform 1 are not supplied |
| Phosphorylation at Ser85, Ser89, Ser91, Ser96, Tyr105 and Ser166 | May alter apparent mobility; no visible shift is demonstrated |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | NCK1 may be depleted from a cytoplasmic fraction after genotoxic stress | Check whole-cell and nuclear fractions and verify antibody performance |
| Band higher than expected | Phosphorylation may affect mobility, but the band identity is unconfirmed | Compare with phosphatase-treated sample and NCK1 depletion |
| Band lower than expected | An isoform or nonspecific signal is possible; isoform masses are unknown | Check with a second NCK1 antibody or NCK1 depletion |
| Multiple bands | Two named isoforms or phosphorylation may contribute, but distinct bands are unproven | Confirm each band with NCK1 depletion |
| Weak or no signal | NCK1 may redistribute from cytoplasm to nucleus after genotoxic stress | Compare whole-cell, cytoplasmic and nuclear samples |
Experiment evidence
Western blot images showing NCK1 band
M02260 · NCK1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for NCK1 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for NCK1 in Western blot, you can use adrenal gland tissue, which shows high expression in HPA.
Positive control: Adrenal gland (IHC candidate; verify WB)
Negative control: Caudate (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: Caudate provides a tissue negative control, but genotoxic stress can shift NCK1 from cytoplasm to nucleus.
HPA tissue expression evidence for NCK1
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 → |
| Bronchus | respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Endometrium | glandular cells | High | Protein (IHC) | HPA → |
| Kidney | collecting ducts | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Caudate | glial cells | Not detected | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | Low | Protein (IHC) | HPA → |
| Hippocampus | neuronal cells | Low | Protein (IHC) | HPA → |
| Skeletal muscle | myocytes | Low | Protein (IHC) | HPA → |
| Skin | keratinocytes | Low | Protein (IHC) | HPA → |
Section 3
Advanced NCK1 Western Blot Tips
Deeper troubleshooting and optimisation questions for NCK1, answered from its protein features.
Where should the main NCK1 band appear?
Band shift · Canonical NCK1 has a predicted mass of 42.9 kDa, consistent with the reported band near 43 kDa. Use the reported position as a guide when identifying the main band.
Could NCK1 isoforms produce different bands?
Isoforms · UniProt lists isoforms 1 and 2. In isoform 2, residues 1–76 of the canonical sequence are replaced by a 12-residue sequence. This could change apparent size and antibody recognition if the antibody binds the altered region; the feature alone does not establish a visible second band.
Which NCK1 modifications matter when assessing a shifted band?
PTM · UniProt lists N-acetylalanine at position 2; phosphoserine at 85, 89, 91, 96 and 166; and phosphotyrosine at 105. These are UniProt canonical-sequence coordinates, which may differ from paper or antibody numbering. Check the antibody’s target region when interpreting a shift; these sites alone do not establish that a shift will be visible.
Does this guide establish induction of NCK1?
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 NCK1?
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 M02260 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 NCK1 be quantified across cellular fractions?
Quantitation · Because NCK1 occurs in the cytoplasm, endoplasmic reticulum and nucleus, keep fraction preparation consistent and compare like fractions. Its predominantly nuclear localization after genotoxic stress can change a fraction’s NCK1 signal without establishing a change in total protein abundance.
How does genotoxic stress affect NCK1 sampling?
Interpretation · NCK1 is mostly cytoplasmic but shuttles into the nucleus through interaction with SOCS7. UniProt reports predominantly nuclear localization after UV irradiation, hydroxyurea or mitomycin C treatment. For these conditions, compare the same cellular fractions across samples; a change in one fraction may reflect redistribution.
First compare them with the reported ~43 kDa band and check whether the antibody can recognize both isoforms, given the isoform 2 replacement of canonical residues 1–76. NCK1 also has listed phosphorylation sites, but their presence alone cannot explain an observed mass difference or confirm the identity of another band.
First compare them with the reported ~43 kDa band and check whether the antibody can recognize both isoforms, given the isoform 2 replacement of canonical residues 1–76. NCK1 also has listed phosphorylation sites, but their presence alone cannot explain an observed mass difference or confirm the identity of another band.
Boster reagents
NCK1 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-Nck NCK1 Monoclonal Antibody
Cat # M02260
The listed anti-NCK1 monoclonal antibody, M02260, is reported to react with human, mouse, and rat. Its Western blot image shows a band near the expected 43 kDa in the specified cell and tissue lysates. Evidence here is limited to that product image.
Which to pick: M02260 is the only listed option. Its Western blot image includes human HeLa, SIHA, A431, and Jurkat cells, plus rat and mouse brain and liver lysates, tested at a 1:500 primary antibody dilution. Choose it when those reported contexts fit your experiment.
Source: BosterBio NCK1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P16333.
- Human Protein Atlas. NCK1 tissue expression.
- PMC4138027 — target-verified WB comparison
- PMC4236421 — target-verified WB comparison
- PMC13486839 — target-verified WB comparison