This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
NEFM / Neurofilament medium polypeptide · Western blot design guide
Design a Western Blot for NEFM
Source-linked NEFM Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-NEFM 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
NEFM 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 | ~102.5 kDa | |
| Observed band | ~160 kDa | |
| Gel | 8% (catalog A06821-2) | |
| Positive control | Caudate (IHC candidate; verify WB) +4 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Phosphorylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Source-Linked NEFM Western Blot Protocol Options
The A06821-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human 293T, human SH-SY5Y, rat brain, mouse brain (catalog A06821-2) |
| Gel % | 8% (catalog A06821-2) |
| Load | 30 ug; reducing conditions (catalog A06821-2) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A06821-2) |
| Membrane | nitrocellulose membrane (catalog A06821-2) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A06821-2) |
| Primary antibody | A06821-2 · 0.5 μg/mL (catalog A06821-2) |
| Primary incubation | overnight at 4°C (catalog A06821-2) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A06821-2) |
| Secondary incubation | 1.5 hour at RT (catalog A06821-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A06821-2) |
| Detection | ECL (catalog A06821-2) |
Section 2
What Is the Expected NEFM Western Blot Band Size?
NEFM is predicted at 102.5 kDa but observed near 160 kDa in antibody QC; the cause of the difference is not established.
| Band near 160 kDa | Empirical NEFM band in reducing lysates; the cause of its migration is unestablished |
| Band near 102.5 kDa | Near the sequence-predicted mass; identity requires confirmation |
| Multiple bands | Isoforms 1 and 2 could contribute, but distinct migration is unproven |
| Single band without a clear glycosylation shift | O-linked GlcNAc at Thr47 and Thr431 does not establish a visible shift |
💡Expected NEFM appearanceUniProt predicts 102.5 kDa, while antibody QC shows a band near 160 kDa in reducing lysates; its higher apparent mass is unexplained, so confirm identity with knockdown or an independent antibody.
| UniProt predicted mass | Sequence-based mass is 102.5 kDa; the observed band is near 160 kDa |
| O-linked GlcNAc at Thr47 | May modify apparent migration; no visible shift is established |
| O-linked GlcNAc at Thr431 | May modify apparent migration; no visible shift is established |
| Isoforms 1 and 2 | Could differ in size, but their relative masses and separation are unknown |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Cytoskeletal NEFM may be poorly recovered from the sampled lysate | Check the insoluble fraction and include a brain lysate positive control |
| Band higher than expected | The observed band near 160 kDa exceeds the 102.5 kDa sequence prediction; the cause is unknown | Compare with the QC band and confirm identity by knockdown or an independent antibody |
| Band lower than expected | An isoform may differ in size, but its mass is unknown | Confirm identity by knockdown or an independent antibody |
| Multiple bands | Isoforms 1 and 2 are annotated, though distinct bands are unproven | Compare bands after knockdown and with an independent antibody |
| Weak or no signal | NEFM is localized to the cytoskeleton, axons and perikarya, so abundance may depend on the sample | Use an appropriate brain or neuronal positive control and check lysate loading |
Experiment evidence
Western blot images showing NEFM band
A06821-2 · NEFM
A06821-2 · NEFM
M06821 · NEFM
M06821 · NEFM
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for NEFM Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for NEFM in Western blot, you can use caudate tissue lysate, where HPA reports high expression.
Positive control: Caudate (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: Caudate is a strong positive choice, while HPA reports no detectable NEFM in adipose tissue.
HPA tissue expression evidence for NEFM
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 |
|---|---|---|---|---|
| Caudate | neuronal cells | High | Protein (IHC) | HPA → |
| Cerebellum | processes in granular layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | neuropil | Medium | Protein (IHC) | HPA → |
| Colon | peripheral nerve/ganglion | Medium | Protein (IHC) | HPA → |
| Placenta | decidual cells | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced NEFM Western Blot Tips
Deeper troubleshooting and optimisation questions for NEFM, answered from its protein features.
How should NEFM 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.
How might the two NEFM isoforms affect band interpretation?
Isoforms · Isoform 2 lacks residues 1–376 of the canonical isoform, so it could yield a distinct band. Do not assign a band to either isoform by apparent size alone. Check whether the antibody recognizes a region present in both isoforms or within the missing segment.
Choose an epitope beyond canonical residue 376 if detection of both isoforms is the goal. Isoform 2 lacks canonical residues 1–376, so an antibody restricted to that segment would miss it. Confirm the antibody’s stated epitope and isoform reactivity before comparing bands.
Choose an epitope beyond canonical residue 376 if detection of both isoforms is the goal. Isoform 2 lacks canonical residues 1–376, so an antibody restricted to that segment would miss it. Confirm the antibody’s stated epitope and isoform reactivity before comparing bands.
Which phosphorylation sites are useful when investigating band changes?
PTM · UniProt annotates phosphorylation at canonical positions Ser-30, Ser-99, Ser-226, Tyr-320, Ser-346, and numerous later sites, including Ser-545, Ser-553, Ser-558, Ser-559, and Thr-571. Compare matched samples with and without phosphatase treatment to test whether phosphorylation contributes to a change. A listed site does not guarantee a visible shift.
Quantify each with an antibody validated for the intended target: total NEFM or a specified phosphorylated site. NEFM has multiple annotated phosphorylation sites, so a signal from one site does not measure phosphorylation across the protein. Compare like bands and like sample conditions, and state which canonical site the phospho measurement addresses.
Quantify each with an antibody validated for the intended target: total NEFM or a specified phosphorylated site. NEFM has multiple annotated phosphorylation sites, so a signal from one site does not measure phosphorylation across the protein. Compare like bands and like sample conditions, and state which canonical site the phospho measurement addresses.
Does this guide establish induction of NEFM?
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 NEFM?
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 A06821-2 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 should NEFM be quantified?
Quantitation · Standard workflow guidance: quantify only a validated target band within the linear exposure range. Use consistent sample preparation and loading, retain biological replicates, and avoid interpreting saturation or loading differences as regulation.
Why does NEFM appear near 160 kDa instead of 102.5 kDa?
Interpretation · 102.5 kDa is the predicted mass, while about 160 kDa is the supplied apparent Western blot band. NEFM has many annotated phosphorylation sites and two O-linked GlcNAc sites, but those features alone do not establish the cause of the difference. Use the observed band as a reference and verify its identity experimentally.
UniProt lists O-linked GlcNAc at threonines 47 and 431 in canonical NEFM. These sites establish that NEFM can be modified, but do not predict a separate visible band or explain the approximately 160 kDa apparent mass. Interpret any additional band with independent evidence.
Consider isoform 2, which lacks canonical residues 1–376, alongside modification-dependent changes or antibody cross-reactivity. NEFM also forms heterodimers with NEFL and INA in vitro, but that annotation alone cannot identify a Western blot band. Check antibody epitope, sample preparation, and band identity before assigning a cause.
UniProt lists O-linked GlcNAc at threonines 47 and 431 in canonical NEFM. These sites establish that NEFM can be modified, but do not predict a separate visible band or explain the approximately 160 kDa apparent mass. Interpret any additional band with independent evidence.
Consider isoform 2, which lacks canonical residues 1–376, alongside modification-dependent changes or antibody cross-reactivity. NEFM also forms heterodimers with NEFL and INA in vitro, but that annotation alone cannot identify a Western blot band. Check antibody epitope, sample preparation, and band identity before assigning a cause.
Boster reagents
NEFM 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-NF-M/NEFM Antibody Picoband®
Cat # A06821-2
Real WB data
Anti-NEFM/Nf M Rabbit Monoclonal Antibody
Cat # M06821
Two the supplier anti-NEFM antibodies have Western blot images. Both captions report a band near 160 kDa, although the expected size is 102 kDa. The images show tested lysates under the stated conditions; they do not establish broader validation.
Which to pick: Choose A06821-2 for the illustrated human cell and rat or mouse brain lysates. M06821 has an image for human 293T and SH-SY5Y lysates; pig and rat are listed as reactive, but neither appears in its image.
Source: BosterBio NEFM gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P07197.
- Human Protein Atlas. NEFM tissue expression.
- PMC6819901 — target-verified WB comparison
- PMC7079284 — target-verified WB comparison
- PMC6054723 — target-verified WB comparison