MLKL / Mixed lineage kinase domain-like protein · Western blot design guide

Design a Western Blot for MLKL

Real validated MLKL Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-MLKL 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 MLKL: expected band ~54.5 kDa, hero antibody M00535-1, catalog values and labelled standard workflow; separate PMC comparisons on the guide — Printable **MLKL** Western blot protocol sheet — expected band ~54.5 kDa, antibody M00535-1, controls and PMC citations. Open the full MLKL WB guide →

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MLKL 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	~54.5 kDa
	Observed band	~54 kDa
	Gel	5–20% (catalog M00535-1)
	Negative control ⓘ	Suggested KO / knockdown lysate

Important caveats

Reasons your observed band may differ from the expected size.

	PTM	Phosphorylated
	Caveat	Phosphorylation-state controls
	Gene-set association	MSigDB C7 membership
	Isoform	2 isoform(s)

Section 1

Real Curated MLKL Western Blot Protocols

The M00535-1 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 Hela, human PC-3, human A549 (catalog M00535-1)
Gel %	5–20% (catalog M00535-1)
Load	30 ug; reducing conditions (catalog M00535-1)
Transfer	a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog M00535-1)
Membrane	nitrocellulose membrane (catalog M00535-1)
Blocking	5% non-fat milk/TBS for 1.5 hour at RT (catalog M00535-1)
Primary antibody	M00535-1 · 1:500 (catalog M00535-1)
Primary incubation	overnight at 4°C (catalog M00535-1)
Secondary antibody	goat anti-rabbit IgG-HRP, 1:5000 (catalog M00535-1)
Secondary incubation	1.5 hour at RT (catalog M00535-1)
Wash	TBS-0.1%Tween 3 times with 5 minutes each (catalog M00535-1)
Detection	ECL (catalog M00535-1)

Section 2

What Is the Expected MLKL Western Blot Band Size?

MLKL is predicted at 54.5 kDa and observed near 54 kDa; the cause of any difference is not established.

What am I looking at on my blot?
Band near 54 kDa in whole-cell lysate	matches the empirical MLKL band
Single sharp band near 54 kDa	consistent with the predicted 54.5 kDa protein
Additional bands at different positions	could reflect isoforms 1 and 2; their migration is not established
More signal in a membrane fraction after necroptosis induction	consistent with MLKL translocation from cytoplasm to plasma membrane

💡Expected MLKL appearanceMLKL has a predicted mass of 54.5 kDa and an empirical band at approximately 54 kDa; confirm band identity with appropriate controls, especially when additional bands appear.

How each factor affects band size
UniProt predicted mass	places the 471-residue protein near 54.5 kDa
Empirical MLKL band	appears at approximately 54 kDa in reducing whole-cell blots
Isoform 1	has no supplied isoform-specific mass or migration
Isoform 2	could differ in size from isoform 1, but its mass and migration are not supplied

Why is my band missing or off?
Situation	Likely cause	Next action
No band in lysate	MLKL expression or detection may be insufficient in the sampled cells	check sample loading and use a validated positive lysate
Band higher than expected	band identity or sample preparation may account for the difference; MLKL forms homotrimers, but their SDS-PAGE migration is not established	repeat under fully denaturing, reducing conditions and verify band identity
Band lower than expected	isoform identity or degradation may account for the difference; isoform masses are not supplied	compare with a validated positive sample and check sample integrity
Multiple bands	isoforms 1 and 2 are annotated, but distinct resolved bands are not established	verify band identity with an independent antibody or MLKL depletion
Weak or no signal	MLKL may be unevenly distributed between cytoplasm and membrane after necroptosis induction	check whole-cell recovery and compare cytoplasmic and membrane fractions

Source: UniProt Q8NB16 Human Protein Atlas product datasheet (M00535-1)

Experiment evidence

Western blot images showing MLKL band

M00535-1 · MLKL

A00535-3 · MLKL

M00535 · MLKL

← swipe / scroll for more images →

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

Sample controls for MLKL Western blot

🧪For positive controls for MLKL in Western blot, you can use an independently validated MLKL-positive sample; no HPA positive tissue or cell line is provided.

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: Without HPA expression data, tissue-based controls cannot be verified from the supplied evidence.

HPA tissue expression evidence for MLKL

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 MLKL Western Blot Tips

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

How should MLKL 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 MLKL isoforms produce different bands?

Isoforms · UniProt lists two isoforms. Isoform 2 replaces residues 179–205 and lacks residues 206–413 relative to the canonical sequence, so it is substantially shorter. A band’s identity depends on which isoforms the antibody recognizes; the sequence difference alone does not establish an apparent band position.

Which MLKL phosphorylation sites should I consider?

PTM · The supplied UniProt coordinates are Ser125, Thr357, Ser358 and Ser360. RIPK3 is specified for Thr357, Ser358 and Ser360; no enzyme is specified for Ser125. Check the numbering convention of any phospho-specific antibody before matching its stated site to these UniProt positions.

MLKL has four listed phosphosites, including three attributed to RIPK3. Their presence alone does not show that phosphorylation causes a visible shift. Assess any shifted band with a site-specific readout or an appropriate phosphorylation control rather than assigning it from apparent mass alone.

How should necroptosis induction affect MLKL blot planning?

Induction · MLKL forms homotrimers and moves from the cytoplasm to the plasma membrane on necroptosis induction. It also forms a complex with PGAM5, RIPK1 and RIPK3 during TNF-induced necrosis. Compare matched conditions and consider cytoplasmic and membrane fractions when assessing redistribution.

How should transfer be checked for MLKL?

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 M00535-1 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.

How can I quantify MLKL across fractions?

Quantitation · MLKL is listed in the cytoplasm and at the cell membrane, with membrane translocation on necroptosis induction. Quantify matched fractions consistently and account for changes in distribution when interpreting a change in either fraction. If measuring total MLKL, include the relevant fractions.

Why is the MLKL band near 54 kDa?

Interpretation · The supplied apparent band is approximately 54 kDa, close to the predicted 54.5 kDa for canonical MLKL. The listed features do not establish a cause for any small difference between calculated and apparent mass.

UniProt reports MLKL homooligomers and homotrimers on necroptosis induction. These features make oligomerization a possibility to investigate, but they do not establish that an unexpected high-mass band is an MLKL oligomer. Compare induction conditions and verify band identity before assigning it.

Boster reagents

MLKL 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 MLKL using anti-MLKL antibody (M00535-1). Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions. Lane 1: human Hela whole cell lysates, Lane 2: human PC-3 whole cell lysates, Lane 3: human A549 whole cell lysates, Lane 4: human MCF-7 whole cell lysates, Lane 5: rat testis tissue lysates, Lane 6: rat liver tissue lysates, Lane 7: mouse testis tissue lysates, Lane 8: mouse liver 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-MLKL antigen affinity purified monoclonal antibody (Catalog # M00535-1) at 1:500 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 Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for MLKL at approximately 54 kDa. The expected band size for MLKL is at 54 kDa.

Anti-MLKL Rabbit Monoclonal Antibody

Cat # M00535-1

View product →

Real WB data

Western blot analysis of MLKL using anti-MLKL antibody (A00535-3). Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions. Lane 1: human PC-3 whole cell lysates, Lane 2: human A549 whole cell lysates, Lane 3: human MCF-7 whole cell lysates, Lane 4: human HepG2 whole cell lysates, Lane 5: rat testis tissue lysates, Lane 6: rat liver tissue lysates, Lane 7: mouse testis tissue lysates, Lane 8: mouse liver 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-MLKL antigen affinity purified polyclonal antibody (Catalog # A00535-3) 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 Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for MLKL at approximately 50 kDa. The expected band size for MLKL is at 54 kDa.

Anti-MLKL Antibody Picoband®

Cat # A00535-3

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Real WB data

Western blot analysis of MLKL using anti-MLKL antibody (M00535). <br>Electrophoresis was performed on a 10% 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. <br>Lane 1: human Hela whole cell lysates,<br>
Lane 2: human PC-3 whole cell lysates,<br>
Lane 3: human A549 whole cell lysates.<br>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-MLKL antigen affinity purified monoclonal antibody (M00535) at 1:500 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 MLKL at approximately 54 kDa. The expected band size for MLKL is at 54 kDa.

Anti-MLKL Rabbit Monoclonal Antibody

Cat # M00535

View product →

Three anti-MLKL antibodies have Western blot images. M00535-1 and A00535-3 show human cell and mouse and rat tissue lysates; M00535 shows human cell lysates. These images document the stated samples and conditions, not performance across every sample type.

Which to pick: For mouse or rat lysates, consider M00535-1 or A00535-3; both have WB images with testis and liver samples. For human cell lysates, all three have images. M00535-1 and M00535 show bands near the expected 54 kDa; A00535-3 shows one near 50 kDa.

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

References

UniProt Consortium. UniProt entry Q8NB16.
Human Protein Atlas. MLKL tissue expression.
PMC6962303 — target-verified WB comparison
PMC9420252 — target-verified WB comparison
PMC8122156 — target-verified WB comparison