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- Table of Contents
CIT / Citron Rho-interacting kinase · Western blot design guide
Design a Western Blot for CIT
Real validated CIT Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CIT 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
CIT 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 | ~231.4 kDa | |
| Observed band | ~231 kDa | |
| Gel | 5–20% (catalog A01331-2) | |
| Positive control | Breast (IHC candidate; verify WB) +4 more | |
| Negative control | Epididymis (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 | 4 isoform(s) |
Section 1
Real Curated CIT Western Blot Protocols
The A01331-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | rat brain, mouse brain (catalog A01331-2) |
| Gel % | 5–20% (catalog A01331-2) |
| Load | 30 ug; reducing conditions (catalog A01331-2) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A01331-2) |
| Membrane | nitrocellulose membrane (catalog A01331-2) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A01331-2) |
| Primary antibody | A01331-2 · 0.5 μg/mL (catalog A01331-2) |
| Primary incubation | overnight at 4°C (catalog A01331-2) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A01331-2) |
| Secondary incubation | 1.5 hour at RT (catalog A01331-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A01331-2) |
| Detection | ECL (catalog A01331-2) |
Section 2
What Is the Expected CIT Western Blot Band Size?
CIT is predicted at 231.4 kDa and observed at ~231 kDa; this agreement needs no additional size explanation, while isoform and homodimer effects remain conditional.
| Band at ~231 kDa | Matches the empirical CIT band and its 231.4 kDa predicted mass |
| Band near ~462 kDa under nonreducing conditions | Could reflect the annotated CIT homodimer |
| Several bands at different positions | Could reflect isoforms 1, 2, 3, and 4 if their migration differs |
| Doublet near ~231 kDa | Could reflect different phosphorylation states, although a mobility difference is unproven |
💡Expected CIT appearanceFull-length CIT has a predicted mass of 231.4 kDa and an empirical band at ~231 kDa; confirm band identity with a validated positive lysate and an independent antibody.
| Predicted CIT mass | Places full-length CIT near the empirical ~231 kDa band |
| CIT homodimer | Could yield a band near twice the monomer mass if the dimer persists during electrophoresis |
| Splice isoforms 1 and 2 | May migrate differently, but their individual masses and separation are unknown |
| Splice isoforms 3 and 4 | May migrate differently, but their individual masses and separation are unknown |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | CIT may be below detection in the sampled lysate | Compare with the rat or mouse brain lysates used in antibody validation |
| Band higher than expected | The annotated homodimer may persist if sample denaturation is incomplete | Compare reducing and nonreducing preparations |
| Band lower than expected | A splice isoform or protein breakdown may be detected | Check with an antibody to another CIT region |
| Multiple bands | Isoforms 1, 2, 3, and 4 may differ in migration, but their band positions are unknown | Compare with an independent CIT antibody or isoform-specific evidence |
| Weak or no signal | Transfer of the large ~231 kDa protein may be inefficient | Check high-molecular-weight transfer and include a validated positive lysate |
Experiment evidence
Western blot images showing CIT band
A01331-2 · CIT
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CIT Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for CIT in Western blot, you can use breast tissue lysate, which HPA scores High.
Positive control: Breast (IHC candidate; verify WB)
Negative control: Epididymis (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: Cytoplasmic CIT can be assessed in tissue lysate, with HPA Not-detected epididymis as a negative control.
HPA tissue expression evidence for CIT
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 |
|---|---|---|---|---|
| Breast | adipocytes | High | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Caudate | neuronal cells | High | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | High | Protein (IHC) | HPA → |
| Esophagus | squamous epithelial cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Epididymis | glandular cells | Not detected | Protein (IHC) | HPA → |
| Pancreas | exocrine glandular cells | Not detected | Protein (IHC) | HPA → |
| Parathyroid gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | smooth muscle cells | Not detected | Protein (IHC) | HPA → |
| Testis | cells in seminiferous ducts | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CIT Western Blot Tips
Deeper troubleshooting and optimisation questions for CIT, answered from its protein features.
How should CIT 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.
Which CIT isoforms could produce smaller bands?
Isoforms · Four isoforms are listed. Isoform 2 lacks canonical residues 483–2027 and should be substantially smaller than full-length CIT. Isoform 3 lacks residues 1–467, 1239–1253, and 1919. Isoform 4 replaces residue 694 with a longer sequence, so its mass may differ from isoform 1.
Compare the antibody’s reported binding region with the UniProt canonical sequence. A region within residues 483–2027 is absent from isoform 2; residues 1–467, 1239–1253, and 1919 are absent from isoform 3. Isoform 2 also changes residues 481–482 from EV to GG, and isoform 4 changes residue 694.
Compare the antibody’s reported binding region with the UniProt canonical sequence. A region within residues 483–2027 is absent from isoform 2; residues 1–467, 1239–1253, and 1919 are absent from isoform 3. Isoform 2 also changes residues 481–482 from EV to GG, and isoform 4 changes residue 694.
Which CIT modifications matter when evaluating band patterns?
PTM · In UniProt canonical coordinates, CIT has N-acetylmethionine at 1; phosphoserine at 433, 440, 480, 582, 1940, and 1993; phosphotyrosine at 1196; N6-acetyllysine at 1721; and phosphothreonine at 2013. Check whether an antibody or paper uses different residue numbering before comparing sites. These annotations alone do not establish a detectable band shift.
Does this guide establish induction of CIT?
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 CIT Western blot?
Transfer · For full-length CIT at about 231 kDa, start with a wet transfer and verify that the high-mass band leaves the gel and reaches the membrane. Adjust transfer conditions if recovery is poor. The shorter annotated isoforms may transfer differently.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A01331-2 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 CIT bands be quantified consistently?
Quantitation · Quantify the same CIT band across samples and record which isoform the antibody can recognize. The approximately 231 kDa band is consistent with full-length CIT, while isoform 2 lacks most of the canonical sequence. Keep distinct bands separate unless their identities are established.
Why might the CIT band differ from its predicted mass?
Interpretation · Full-length CIT has a predicted mass of 231.4 kDa, close to the reported apparent band near 231 kDa. CIT has annotated modifications and splice variants, but their presence alone does not establish a visible shift or explain any mass difference.
Check apparent size and antibody binding region against the four annotated isoforms first. Isoform 2 lacks residues 483–2027; isoform 3 has three deletions; isoform 4 has an insertion at canonical residue 694. CIT also has annotated modifications, but these features alone cannot identify an unexpected band or prove a mobility shift.
Check apparent size and antibody binding region against the four annotated isoforms first. Isoform 2 lacks residues 483–2027; isoform 3 has three deletions; isoform 4 has an insertion at canonical residue 694. CIT also has annotated modifications, but these features alone cannot identify an unexpected band or prove a mobility shift.
Boster reagents
CIT 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-CIT Antibody Picoband®
Cat # A01331-2
The catalog reports one anti-CIT antibody for Western blot, A01331-2. Its WB image shows a band near 231 kDa in rat and mouse brain lysates. Human reactivity is listed, but the supplied image does not show a human sample.
Which to pick: A01331-2 is the only listed option. Its WB example uses rat and mouse brain lysates, 30 µg per lane, and antibody at 0.5 µg/mL. For human samples, the listed reactivity has no accompanying WB image here.
Source: BosterBio CIT gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O14578.
- Human Protein Atlas. CIT tissue expression.
- PMC5641103 — target-verified WB comparison
- PMC7060418 — target-verified WB comparison