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- Table of Contents
CALU / Calumenin · Western blot design guide
Design a Western Blot for CALU
Real validated CALU Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CALU 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
CALU 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 | ~37.1 kDa | |
| Observed band | ≈47 kDa | |
| Gel | 10% (catalog M03193-1) | |
| 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 | Glycosylated + Phosphorylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 15 isoform(s) |
Section 1
Real Curated CALU Western Blot Protocols
The M03193-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human U251, human K562, rat RH35 (catalog M03193-1) |
| Gel % | 10% (catalog M03193-1) |
| Load | 30 ug; reducing conditions (catalog M03193-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog M03193-1) |
| Membrane | nitrocellulose membrane (catalog M03193-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog M03193-1) |
| Primary antibody | M03193-1 · 1:500 (catalog M03193-1) |
| Primary incubation | overnight at 4°C (catalog M03193-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog M03193-1) |
| Secondary incubation | 1.5 hour at RT (catalog M03193-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog M03193-1) |
| Detection | ECL (catalog M03193-1) |
Section 2
What Is the Expected CALU Western Blot Band Size?
CALU is predicted at 37.1 kDa, but an approximately 47 kDa band was observed in reducing whole-cell blots; the cause of the difference is not established.
| Band near 47 kDa | Empirical CALU band in reducing whole-cell lysates; the cause of its migration above the predicted mass is unestablished |
| Band near 37.1 kDa | Near the predicted precursor mass; confirm its identity |
| Band below the precursor position | Signal-peptide cleavage may contribute; confirm the band’s identity |
| Higher or variable band position | Complex N-linked glycosylation at Asn131 may affect migration, but a visible shift is unproven |
| Several bands | CALU has 15 named isoforms, though distinct band positions are unestablished |
| Weak or absent lysate band | Secreted CALU may be depleted from the sampled cells |
💡Expected CALU appearanceUniProt predicts a 37.1 kDa precursor, while reducing whole-cell blots show an approximately 47 kDa band; the difference is unexplained, so confirm band identity with ordinary controls.
| UniProt predicted precursor mass | 37.1 kDa sequence-based reference; the empirical band is approximately 47 kDa |
| Complex N-linked glycosylation at Asn131 | May affect apparent migration; its contribution to the 47 kDa band is unestablished |
| Signal peptide at residues 1–19 | Cleavage yields a shorter mature chain, without an established band position |
| Isoforms 1–15 | Alternative sequences may differ in size; individual masses and resolved band positions are unavailable |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | CALU is secreted and may be depleted from sampled cells | Compare matched whole-cell lysate and conditioned medium with a positive control |
| Band higher than expected | The observed 47 kDa band exceeds the 37.1 kDa prediction; its cause is unestablished | Check band identity with an independent CALU antibody or CALU depletion; assess glycosylation with a paired deglycosylated sample |
| Band lower than expected | Signal-peptide cleavage may shorten CALU | Confirm identity with an independent antibody or CALU depletion before assigning cleavage |
| Broad smear instead of sharp band | Variable processing of the complex N-linked site at Asn131 is possible | Compare treated and untreated samples after N-glycan removal; verify CALU identity |
| Multiple bands | CALU has 15 named isoforms, but their band positions are unknown | Use an independent antibody or CALU depletion to determine which bands are CALU |
Experiment evidence
Western blot images showing CALU band
M03193-1 · CALU
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CALU Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for CALU in Western blot, you can use adrenal gland tissue, which HPA rates as highly expressed.
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 the samples.
⚠️Feasibility: HPA lists no tissue as not detected, so use siRNA knockdown or a KO line for a negative control.
HPA tissue expression evidence for CALU
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 → |
| Breast | glandular cells | High | Protein (IHC) | HPA → |
| Caudate | glial cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | glial cells | High | Protein (IHC) | HPA → |
| Endometrium | glandular cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | adipocytes | Low | Protein (IHC) | HPA → |
| Appendix | glandular cells | Low | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Low | Protein (IHC) | HPA → |
| Duodenum | glandular cells | Low | Protein (IHC) | HPA → |
| Nasopharynx | respiratory epithelial cells | Low | Protein (IHC) | HPA → |
Section 3
Advanced CALU Western Blot Tips
Deeper troubleshooting and optimisation questions for CALU, answered from its protein features.
How should CALU 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 CALU isoforms produce different bands?
Isoforms · UniProt lists 15 isoforms with substantial sequence differences. For example, isoform 15 lacks residues 1–151, while isoform 9 lacks 94–254. Check which isoforms your antibody can recognize before assigning additional bands to CALU.
Match its recognized region to the isoforms of interest. Isoform 12 lacks residues 75–315, and isoform 15 lacks 1–151; other isoforms have substitutions or shorter deletions. An antibody against a missing region may fail to detect that isoform.
Match its recognized region to the isoforms of interest. Isoform 12 lacks residues 75–315, and isoform 15 lacks 1–151; other isoforms have substitutions or shorter deletions. An antibody against a missing region may fail to detect that isoform.
Which CALU modifications matter when assessing band changes?
PTM · UniProt lists N-linked glycosylation at Asn131; phosphorylation at Ser44, Tyr47, Thr65, Ser69 by FAM20C, Thr254, Ser261, and Ser277; and acetylation at Lys165. These are canonical UniProt coordinates and may differ from paper or antibody numbering. The annotations do not establish that any modification produces a visible shift.
Does this guide establish induction of CALU?
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 CALU?
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 M03193-1 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.
What should be quantified when CALU shows several bands?
Quantitation · Specify the band or set of bands measured and use that choice consistently across samples. Because CALU has 15 annotated isoforms, band intensity cannot automatically be treated as one isoform’s abundance; confirm that the antibody recognizes the isoforms included in the measurement.
Why might CALU appear near 47 kDa instead of 37.1 kDa?
Interpretation · The predicted mass is 37.1 kDa, while the reported apparent band is about 47 kDa. CALU has a 1–19 signal peptide and an N-linked glycosylation site at Asn131. These features are relevant when assessing mobility, but their presence alone does not establish the cause or size of the difference.
CALU has a signal peptide at residues 1–19. Its removal would shorten the protein, so that feature alone does not explain an apparent band near 47 kDa versus the 37.1 kDa predicted mass.
Compare each band with the antibody’s recognized region and the listed isoform sequences. CALU has 15 annotated isoforms, a 1–19 signal peptide, and glycosylation and modified-residue annotations. These features offer possibilities to investigate, but they do not identify any particular unexpected band.
CALU has a signal peptide at residues 1–19. Its removal would shorten the protein, so that feature alone does not explain an apparent band near 47 kDa versus the 37.1 kDa predicted mass.
Compare each band with the antibody’s recognized region and the listed isoform sequences. CALU has 15 annotated isoforms, a 1–19 signal peptide, and glycosylation and modified-residue annotations. These features offer possibilities to investigate, but they do not identify any particular unexpected band.
Boster reagents
CALU 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-Calumenin Rabbit Monoclonal Antibody
Cat # M03193-1
The catalog reports one anti-CALU antibody for Western blot: M03193-1. Its validation image shows human U251 and K562, rat RH35, and mouse HEPA1-6 lysates. The reported band is approximately 47 kDa, versus an expected 37 kDa; the supplied evidence does not resolve this difference.
Which to pick: M03193-1 is the only listed option. It reports human, mouse, and rat reactivity and has a Western blot image from the named cell lysates. Check whether the observed 47 kDa band fits your experiment.
Source: BosterBio CALU gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O43852.
- Human Protein Atlas. CALU tissue expression.
- PMC7259615 — target-verified WB comparison
- PMC11439346 — target-verified WB comparison