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CRLF2 · Western blot design guide
Design a Western Blot for CRLF2
Real validated CRLF2 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CRLF2 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
CRLF2 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 | ~42 kDa | |
| Observed band | 65-75 kDa | |
| Gel | 10–12% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Cleaved | |
| Caveat | Heavy N-glycosylation | |
| Regulation | IL-6 up | |
| Isoform | 3 isoform(s) |
Section 1
Real Curated CRLF2 Western Blot Protocols
Literature-validated Western blot parameters for CRLF2 — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Gel % | 10–12% |
| Transfer | nitrocellulose membrane, 150 mA, 50–90 min |
| Membrane | nitrocellulose |
| Blocking | 5% non-fat milk / TBS, 1.5 h RT |
| Primary antibody | 0.5 µg/mL |
| Primary incubation | overnight at 4 °C |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:10000 |
| Wash | TBS-0.1% Tween, 3 × 5 min |
| Detection | ECL |
| Exposure / imaging | Tanon 5200 |
| Observed band | 65–75 kDa |
Section 2
What Is the Expected CRLF2 Western Blot Band Size?
CRLF2 has a 42 kDa predicted backbone but runs at 65-75 kDa on Western blots due to heavy N-linked glycosylation at four asparagine sites.
| band around 65-75 kDa | mature, N-glycosylated CRLF2 migrating well above the 42 kDa predicted mass because of glycan addition at 4 N-linked sites |
| diffuse or smeared band rather than one sharp line | heterogeneous glycan occupancy across the 4 N-linked sites produces a range of glycoform masses that run close together |
| band closer to 40-42 kDa | unglycosylated or enzymatically deglycosylated core protein running near the predicted backbone mass |
| mature band slightly smaller than the full-length precursor | cleavage of the signal peptide (residues 1-22) removes a small amount of mass to generate the membrane-bound mature protein |
| little or no signal in standard whole-cell lysate | CRLF2 is annotated as both a membrane receptor and a secreted form, so a shed/soluble pool may be lost if only whole-cell lysate is probed |
| more than one distinct band across the lane | three annotated splice isoforms of CRLF2 can differ in length and appear as separate bands |
💡Expected CRLF2 appearanceCRLF2 is predicted at 42 kDa, but the mature, N-glycosylated receptor typically appears as a broader band around 65-75 kDa by Western blot, reflecting glycosylation at four N-linked sites and signal peptide cleavage.
| Predicted mass (UniProt) | the unmodified 371-aa backbone calculates to 42 kDa, well below what is typically observed |
| N-glycosylation at Asn47, Asn55, Asn101, Asn169 | four N-linked glycans add substantial mass, shifting the mature band up into the observed 65-75 kDa range |
| Signal peptide cleavage (residues 1-22) | removal of the signal sequence trims mass from the full-length precursor to give the mature membrane-bound form |
| Alternative splicing (isoforms 1, 2, 3) | different isoforms vary in length and can appear as additional bands of differing relative size on the same blot |
| Non-glycosylated recombinant standard | bacterially expressed or otherwise non-glycosylated recombinant CRLF2 runs closer to the 42 kDa predicted mass, well below native lysate bands |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | heavy N-glycosylation across four sites shifts the mature receptor well above the 42 kDa predicted mass | compare the band against the expected 65-75 kDa range rather than the calculated mass, and confirm with a PNGase F deglycosylation control |
| Broad smear instead of sharp band | heterogeneous glycan occupancy across the four N-linked sites produces a spread of apparent masses | run a lower-percentage gel with a longer separation time, or deglycosylate the sample before loading to sharpen the band |
| No band in lysate | CRLF2 is a membrane receptor with a secreted form annotated, so standard whole-cell lysis may not capture a shed soluble pool or may under-solubilize the membrane fraction | use a membrane-protein-compatible lysis buffer and check conditioned media or secreted fractions alongside whole-cell lysate |
| Multiple bands | three annotated splice isoforms can generate bands distinct from the primary mature receptor | confirm which isoform(s) the antibody epitope targets and compare the banding pattern against isoform-specific expectations |
| Band lower than expected | a non-glycosylated recombinant standard, such as a bacterially expressed protein, lacks the N-linked glycans present on the native receptor | confirm the sample source and compare the recombinant standard against native lysate, which should run higher due to glycosylation |
| Fragments below expected size | proteolytic degradation of the membrane-embedded receptor during lysis and handling | include protease inhibitors and keep samples cold throughout lysis and downstream handling |
Experiment evidence
Western blot images showing CRLF2 band
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CRLF2 Western blot
🧪For positive controls for CRLF2 in Western blot, you can use a CRLF2-transfected/overexpressing cell line, since no tissue- or cell-line-specific expression data was available to identify an endogenous positive source.
Positive control: CRLF2-overexpressing cell line
Negative control: parental (non-transfected) cell line; use siRNA knockdown or KO
Loading controls: Run GAPDH and β-actin alongside a total-protein stain (e.g., stain-free gel, Ponceau S, or REVERT) as loading controls.
⚠️Feasibility: With no HPA expression data available, an endogenous tissue-based positive control cannot be confirmed, so an overexpression system paired with siRNA/KO validation is the most reliable approach; as a single-pass type I membrane protein with a reported secreted/extracellular form, use a membrane-protein-compatible lysis buffer and consider conditioned medium if probing for the secreted species.
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.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Section 3
Advanced CRLF2 Western Blot Tips
Deeper troubleshooting and optimisation questions for CRLF2, answered from its protein features.
Why does CRLF2 run heavier than its predicted mass?
CRLF2's predicted mass is 42 kDa, but it carries 4 N-glycosylation sites, adding substantial carbohydrate mass. This shifts the observed band to 65-75 kDa. Confirm identity with PNGase F deglycosylation, which should shift the band closer to 42 kDa if glycosylation is the cause.
Which isoform is detected in membrane preparations?
Only isoform 1 is annotated as the cell-membrane, single-pass type I protein; isoforms 2 and 3 arise from alternative splicing and may lack the transmembrane region. If probing lysates without membrane enrichment, verify which isoform your antibody epitope targets, since splice variants can differ in apparent size.
Does glycosylation affect CRLF2 detection consistency?
CRLF2 has no annotated phosphorylation or other modified residues; its only listed PTM is N-glycosylation across 4 sites. Because glycosylation efficiency can vary by cell type and treatment, band intensity and position may shift between samples. Treat lysates with PNGase F to normalize glycoform heterogeneity before comparing across conditions.
What blocking buffer avoids interference for CRLF2?
Because CRLF2 is a glycoprotein, milk-based blockers containing glycoproteins can cause lectin-like background from carbohydrate-binding contaminants. Use a BSA-based blocking buffer instead to reduce nonspecific signal near the 65-75 kDa region where CRLF2 migrates.
What transfer method to use for CRLF2 Western blot?
CRLF2 is a single-pass type I membrane glycoprotein with 2 disulfide bonds, so use wet transfer with reduced methanol (around 10%) to preserve efficient elution of this large, heavily glycosylated receptor. Extend transfer time given its membrane-embedded, heterodimeric context with IL7R.
How should CRLF2 signal be normalized for quantitation?
Since CRLF2 functions as a heterodimer with IL7R, its expression can track with IL7R levels rather than general housekeeping genes. Use total protein normalization instead of a single cytosolic loading control, as membrane receptor abundance varies independently of standard housekeeping proteins.
Why might extra bands appear above 75 kDa?
CRLF2 forms 2 disulfide bonds and heterodimerizes with IL7R; under non-reducing conditions, disulfide-linked dimers or the CRLF2-IL7R complex can appear as higher-molecular-weight bands. Always run samples under reducing conditions (DTT or beta-mercaptoethanol) to resolve monomeric CRLF2 at its glycosylated 65-75 kDa size.
Boster reagents
Best CRLF2 Western Blot Antibodies
BosterBio's CRLF2 antibodies are among the best-performing WB antibodies on the market — well cited, thoroughly validated, and orthogonally cross-validated against negative tissues and complementary methods.
Real WB data
Anti-CRLF2 Antibody Picoband®
Cat # A04167-1
For CRLF2 Western blots, we recommend Boster's top-performing anti-CRLF2 antibody, extensively validated and widely cited, with specificity confirmed through orthogonal testing against negative tissue and complementary detection methods for reliable, reproducible results.
Which to pick: Only one Boster anti-CRLF2 antibody is catalogued, A04167-1, so it's the clear choice here — it includes an actual Western blot validation image showing detection of recombinant CRLF2 protein, supporting confidence in its WB performance.
Source: BosterBio CRLF2 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9HC73.
- Human Protein Atlas. CRLF2 tissue expression.