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- Table of Contents
CRH / Corticoliberin · Western blot design guide
Design a Western Blot for CRH
Real validated CRH Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CRH 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
CRH 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 | ~21.4 kDa | |
| Observed band | ≈25 kDa | |
| Gel | 5–20% (catalog A00629-4) | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Cleaved | |
| Caveat | Precursor versus mature peptide | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated CRH Western Blot Protocols
The A00629-4 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human SH-SY5Y, human U-87MG, human U251 (catalog A00629-4) |
| Gel % | 5–20% (catalog A00629-4) |
| Load | 30 ug; reducing conditions (catalog A00629-4) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A00629-4) |
| Membrane | nitrocellulose membrane (catalog A00629-4) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A00629-4) |
| Primary antibody | A00629-4 · 0.5 μg/mL (catalog A00629-4) |
| Primary incubation | overnight at 4°C (catalog A00629-4) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A00629-4) |
| Secondary incubation | 1.5 hour at RT (catalog A00629-4) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A00629-4) |
| Detection | ECL (catalog A00629-4) |
Section 2
What Is the Expected CRH Western Blot Band Size?
CRH is predicted at 21.4 kDa, while antibody QC observes approximately 25 kDa; the cause of this difference is not established.
| Band near 25 kDa | Empirical CRH-reactive band; confirm identity with antibody controls |
| Band near 21.4 kDa | Possible full-length precursor near its predicted mass |
| Band below the precursor | Possible product of signal-peptide or propeptide cleavage |
| Little or no band in whole-cell lysate | CRH is secreted |
💡Expected CRH appearanceThe full-length CRH precursor has a predicted mass of 21.4 kDa, while antibody QC shows a band near 25 kDa; the cause of the difference is unestablished, so confirm identity with antibody controls.
| Predicted precursor mass | 21.4 kDa predicted; the empirical CRH-reactive band is near 25 kDa |
| Signal peptide at residues 1–24 | Cleavage can yield a product smaller than the full-length precursor |
| Propeptide at residues 25–153 | Processing can yield a product smaller than the full-length precursor |
| Cleavage on pairs of basic residues | Processing can produce smaller fragments; their apparent masses are not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | CRH is secreted | Check conditioned medium and a positive-control lysate |
| Band higher than expected | The observed 25 kDa band exceeds the 21.4 kDa prediction for an unestablished reason | Confirm band identity with an independent antibody or CRH-depleted control |
| Band lower than expected | Signal-peptide or propeptide cleavage may produce a smaller form | Check whether the antibody recognizes the processed region and compare appropriate controls |
| Fragments below expected size | Propeptide processing may produce smaller fragments | Check antibody epitope coverage and confirm fragments with an independent antibody |
| Weak or no signal | Secretion may leave little CRH in the sampled cells | Test conditioned medium alongside a positive control |
Experiment evidence
Western blot images showing CRH band
A00629-4 · CRH
A00629-2 · CRH
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CRH Western blot
🧪For positive controls for CRH in Western blot, you can use an HPA-supported positive sample once one is identified; none is supplied here.
Positive control: No high/medium HPA tissue identified
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: CRH is secreted, so conditioned medium may give a stronger signal than whole-cell lysate.
HPA tissue expression evidence for CRH
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 |
|---|---|---|---|---|
| No high/medium HPA tissues identified in the supplied evidence. | ||||
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| No lower-expression tissue rows available in the supplied evidence. | ||||
Section 3
Advanced CRH Western Blot Tips
Deeper troubleshooting and optimisation questions for CRH, answered from its protein features.
Could CRH processing produce bands at different sizes?
Band shift · CRH has a signal peptide at residues 1–24 and a propeptide at 25–153; UniProt also flags cleavage on paired basic residues. Antibodies recognizing different regions may detect different processed forms. Check the antibody epitope before assigning a band to a precursor or processed product.
Are alternative CRH isoforms expected on this blot?
Isoforms · The supplied UniProt record lists one isoform and no alternative sequence. Do not assign multiple bands to annotated splice isoforms on this evidence alone; assess processing and antibody specificity first.
Does CRH have a modification relevant to band interpretation?
PTM · UniProt lists isoleucine amide at position 194, using its 196-residue sequence numbering. Keep that coordinate when comparing antibody or paper annotations, which may use another numbering convention. The listed amidation does not establish a visible band shift.
Does this guide establish induction of CRH?
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 CRH?
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 A00629-4 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 secreted CRH be quantified by Western blot?
Quantitation · UniProt identifies CRH as secreted and lists a signal peptide and propeptide. Choose the sample compartment and antibody epitope to match the CRH form being measured. Quantify bands assigned to the same form consistently; a total across differently processed bands needs a stated rationale.
Why might CRH appear near 25 kDa instead of 21.4 kDa?
Interpretation · The reported apparent band is approximately 25 kDa, while the predicted mass is 21.4 kDa. UniProt lists a signal peptide at residues 1–24 and a propeptide at 25–153, but those features alone do not explain the difference. Treat the band assignment as provisional and check which region the antibody recognizes.
First check the antibody epitope against the signal peptide (1–24), propeptide (25–153), and remaining sequence. Processing could affect which forms are detectable, but the listed features do not identify any particular unexpected band. The record lists no glycosylation sites or alternative sequences to support those assignments.
First check the antibody epitope against the signal peptide (1–24), propeptide (25–153), and remaining sequence. Processing could affect which forms are detectable, but the listed features do not identify any particular unexpected band. The record lists no glycosylation sites or alternative sequences to support those assignments.
Boster reagents
CRH 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-CRF/CRH Antibody Picoband®
Cat # A00629-4
Real WB data
Anti-Corticoliberin CRH Antibody
Cat # A00629-2
Both listed anti-CRH antibodies have Western blot images. A00629-4 was shown with human, rat, and mouse lysates, with a reported band near 25 kDa versus 21 kDa expected. A00629-2 was shown with human placenta lysate. Evidence is limited to these examples.
Which to pick: Choose A00629-4 for the reported human cell, rat, or mouse lysate contexts; choose A00629-2 for the reported human placenta context. Both have WB images, but the supplied evidence does not establish performance in other specimens.
Source: BosterBio CRH gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P06850.
- Human Protein Atlas. CRH tissue expression.
- PMC7005524 — target-verified WB comparison
- PMC6664053 — target-verified WB comparison
- PMC9061884 — target-verified WB comparison