NID1 / Nidogen-1 · Western blot design guide

Design a Western Blot for NID1

Source-linked NID1 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-NID1 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

Western blot protocol sheet for NID1: expected band ~136.4 kDa, hero antibody A05621, catalog values and labelled standard workflow; separate PMC comparisons on the guide — Printable **NID1** Western blot protocol sheet — expected band ~136.4 kDa, antibody A05621, controls and PMC citations. Open the full NID1 WB guide →

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NID1 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	~136.4 kDa
	Observed band	~150 kDa
	Gel	8% (catalog A05621)
	Negative control ⓘ	Suggested KO / knockdown lysate

Important caveats

Reasons your observed band may differ from the expected size.

	PTM	Glycosylated + Cleaved
	Caveat	Modification-state controls
	Gene-set association	MSigDB C7 membership
	Isoform	2 isoform(s)

Section 1

Source-Linked NID1 Western Blot Protocol Options

The A05621 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.

Recommended Western blot protocol parameters
Sample / lysate	human HepG2, human THP-1, human 293T, mouse heart (catalog A05621)
Gel %	8% (catalog A05621)
Load	30 ug; reducing conditions (catalog A05621)
Transfer	a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A05621)
Membrane	nitrocellulose membrane (catalog A05621)
Blocking	5% non-fat milk/TBS for 1.5 hour at RT (catalog A05621)
Primary antibody	A05621 · 0.5 μg/mL (catalog A05621)
Primary incubation	overnight at 4°C (catalog A05621)
Secondary antibody	goat anti-rabbit IgG-HRP, 1:5000 (catalog A05621)
Secondary incubation	1.5 hour at RT (catalog A05621)
Wash	TBS-0.1%Tween 3 times with 5 minutes each (catalog A05621)
Detection	ECL (catalog A05621)

Section 2

What Is the Expected NID1 Western Blot Band Size?

NID1 is predicted at 136.4 kDa and observed near 150 kDa in antibody QC blots; the cause of the difference is not established.

What am I looking at on my blot?
Band near 150 kDa	Empirical NID1 band in antibody QC blots; its difference from the 136.4 kDa predicted mass has no established cause.
Little or no band in whole-cell lysate	NID1 is secreted into the extracellular matrix.
Band below the precursor	Signal-peptide cleavage could produce a smaller mature protein; confirm its identity.
Multiple bands	Isoforms 1 and 2 are annotated, but distinct migration has not been demonstrated.

💡Expected NID1 appearanceUniProt predicts 136.4 kDa, while antibody QC detects a band near 150 kDa; the difference is unexplained, so confirm band identity with appropriate controls.

How each factor affects band size
UniProt predicted mass	136.4 kDa is the precursor sequence mass; the empirical band is near 150 kDa.
O-linked GalNAc at Thr922	May affect migration, but a visible shift is not established.
O-linked GalNAc at Thr935	May affect migration, but a visible shift is not established.
Signal peptide at residues 1–28	Cleavage makes the mature protein smaller than the precursor; its apparent mass is not supplied.
Isoforms 1 and 2	Sequence differences may affect size, but their relative masses and migration are not supplied.

Why is my band missing or off?
Situation	Likely cause	Next action
No band in lysate	NID1 is secreted into the extracellular matrix.	Check conditioned medium or an extracellular matrix fraction alongside a positive control.
Band higher than expected	The observed band near 150 kDa exceeds the 136.4 kDa prediction; the cause is unestablished.	Confirm identity with a positive control and NID1 depletion.
Band lower than expected	Signal-peptide cleavage can lower mass relative to the precursor.	Compare with a positive control and verify band identity before assigning processing.
Multiple bands	Isoforms 1 and 2 are annotated, but their migration is unknown.	Check isoform expression and use NID1 depletion to identify specific bands.
Weak or no signal	Secreted NID1 may be scarce in the sampled cell fraction.	Test conditioned medium or extracellular matrix material with a positive control.

Source: UniProt P14543 Human Protein Atlas product datasheet (A05621)

Experiment evidence

Western blot images showing NID1 band

A05621 · NID1

M05621 · NID1

A05621-1 · NID1

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Real WB data behind the band-size expectations in this section.

Sample controls for NID1 Western blot

🧪For positive controls for NID1 in Western blot, you can use a confirmed NID1-positive basement-membrane sample; the supplied HPA evidence identifies no positive tissue or cell.

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 such as Ponceau alongside the samples.

⚠️Feasibility: Because NID1 is secreted into the extracellular matrix, whole-cell lysates may give weak signal; an extracellular-matrix-enriched sample may work better.

HPA tissue expression evidence for NID1

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 NID1 Western Blot Tips

Deeper troubleshooting and optimisation questions for NID1, answered from its protein features.

Does signal peptide removal explain NID1 band size?

Band shift · The canonical sequence includes a signal peptide at residues 1–28, consistent with NID1 being secreted. Processing can change the sequence present in mature protein, but this feature alone does not explain why the observed band is approximately 150 kDa rather than the 136.4 kDa predicted mass.

How could NID1 isoforms affect band interpretation?

Isoforms · NID1 has isoforms 1 and 2. Isoform 2 lacks canonical residues 710–842, so a lower band could reflect isoform 2. Check whether the antibody target lies within that missing region before comparing isoform bands.

Which NID1 modifications should be considered when interpreting bands?

PTM · UniProt lists sulfotyrosines at residues 289 and 296 and O-linked GalNAc glycosylation at threonines 922 and 935. These are UniProt canonical coordinates; paper or antibody numbering may differ. Their presence does not establish a visible band shift.

Does this guide establish induction of NID1?

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 NID1 Western blot?

Transfer · NID1 is a large protein, with a predicted mass of 136.4 kDa and an observed band near 150 kDa. Choose transfer conditions that retain and transfer proteins around 150 kDa, then check transfer efficiency in that region. The supplied features do not identify a specific transfer method.

How should blocking be optimized?

Blocking · Standard workflow guidance: follow the A05621 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.

Which sample fraction is relevant for NID1 quantitation?

Quantitation · NID1 is secreted and located in the extracellular space, extracellular matrix, and basement membrane. Compare like sample fractions across conditions; a cell lysate alone may not represent total NID1. Quantify a consistently identified band and account for the two isoforms when interpreting changes.

Why might NID1 migrate near 150 kDa instead of 136.4 kDa?

Interpretation · The observed band is approximately 150 kDa, while the predicted mass is 136.4 kDa. NID1 has a signal peptide at residues 1–28 and listed glycosylation and sulfation sites. These features alone do not establish the cause of the apparent mass difference; compare the band with an appropriate molecular weight marker.

Consider the two isoforms, including isoform 2's deletion of residues 710–842, and whether the antibody recognizes that region. NID1 also has 22 listed disulfide bonds, so compare samples prepared under the same reducing conditions. These features suggest checks, but do not identify an unexpected band by themselves.

Boster reagents

NID1 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

Western blot analysis of NID1 using anti-NID1 antibody (A05621). Electrophoresis was performed on a 8% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions. Lane 1: human HepG2 whole cell lysates, Lane 2: human THP-1 whole cell lysates, Lane 3: human 293T whole cell lysates, Lane 4: mouse heart tissue lysates. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-NID1 antigen affinity purified polyclonal antibody (A05621) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an ECL Plus Western Blotting Substrate (Catalog # AR1196-200) with Tanon 5200 system. A specific band was detected for NID1 at approximately 150 kDa. The expected band size for NID1 is at 136 kDa.

Anti-Entactin/NID1 Antibody Picoband®

Cat # A05621

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Real WB data Western blot analysis of Nidogen 1 expression in Human fetal heart lysate.

Anti-Nidogen 1 Rabbit Monoclonal Antibody

Cat # M05621

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Real WB data Western blot analysis of lysates from K562 cells, using NID1 Antibody. The lane on the right is blocked with the synthesized peptide.

Anti-NID1/Entactin Antibody

Cat # A05621-1

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Three the supplier anti-NID1 antibodies have WB images: A05621 in human cell lines and mouse heart, M05621 in human fetal heart, and A05621-1 in K562 cells with peptide blocking. These captions document specific samples, not broad validation.

Which to pick: Choose A05621 for its documented human and mouse WB samples and detailed conditions. M05621 shows human fetal heart lysate. A05621-1 lists human, mouse, and rat reactivity, but its WB image uses K562 cells; rat WB performance is not shown.

Source: BosterBio NID1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.

References

UniProt Consortium. UniProt entry P14543.
Human Protein Atlas. NID1 tissue expression.
PMC8034893 — target-verified WB comparison
PMC11558908 — target-verified WB comparison
PMC11988693 — target-verified WB comparison