erlin1 Antibody

What is the optimal application for ERLIN1 antibody detection in cellular research?

Western blotting represents the most validated application for ERLIN1 antibody detection, with robust performance at dilutions ranging from 1:1000-1:6000. For optimal results, researchers should use ER membrane-enriched fractions rather than whole cell lysates, as ERLIN1 antibodies can cross-react with several unrelated proteins in total cell lysates. This issue can be ameliorated by analyzing membrane preparations, which significantly improves specificity. For immunohistochemistry applications, a dilution range of 1:50-1:500 is recommended with suggested antigen retrieval using TE buffer at pH 9.0 or alternatively citrate buffer at pH 6.0 .

How can I distinguish between ERLIN1 and ERLIN2 in experimental systems?

Distinguishing between ERLIN1 and ERLIN2 requires careful antibody selection as these proteins share significant homology. ERLIN1 has an observed molecular weight of 39-41 kDa, while ERLIN2 runs at approximately 43 kDa on SDS-PAGE. When using pan-ERLIN antibodies (which recognize conserved regions of both proteins), researchers should note that ERLIN2 is typically more abundant than ERLIN1 in most cell types with an approximate ratio of 2:1 (ERLIN2:ERLIN1). For definitive identification, use antibodies raised against non-conserved regions or consider immunoprecipitation with specific antibodies followed by mass spectrometry analysis .

What control samples should be included when validating a new ERLIN1 antibody?

When validating a new ERLIN1 antibody, include the following controls:

• Positive control tissues/cells: HL-60 cells, COLO 320 cells, human kidney tissue, human ovary cancer tissue, rat pancreas tissue, human brain tissue, mouse liver tissue, U-251 cells, or U-397 cells have all shown reliable ERLIN1 expression

• Negative controls: ERLIN1 knockout (E1KO) cells provide the most stringent negative control

• Peptide blocking: Pre-incubation with the immunogen peptide should abolish specific signals

• Molecular weight verification: ERLIN1 should appear at approximately 39-41 kDa

• Cross-reactivity assessment: Test across multiple species if cross-species reactivity is claimed

How do mutations in ERLIN1 affect antibody detection and experimental interpretations?

Mutations in ERLIN1, particularly those associated with hereditary spastic paraplegia, can significantly alter antibody binding and experimental outcomes. The T65I mutation in ERLIN2 (analogous to mutations found in ERLIN1) has been shown to disrupt the formation of functional ERLIN1/2 complexes, preventing the binding of RNF170 and IP3R1 ubiquitination. When working with patient-derived samples or mutant constructs, researchers should employ multiple antibodies targeting different epitopes and combine immunological techniques with functional assays. Western blotting alone may not reveal functional deficits in mutant ERLIN1 proteins that maintain structural integrity but lose interaction capabilities .

What are the critical considerations when studying ERLIN1 in the context of lipid raft dynamics and cholesterol homeostasis?

When investigating ERLIN1 in lipid raft dynamics and cholesterol homeostasis, researchers must consider several methodological factors:

• Membrane fractionation: ERLIN1/2 complexes accumulate in detergent-resistant membranes (DRMs) after solubilization with non-ionic detergent (Triton X-100) and can be isolated by flotation using Optiprep gradients

• Lipid binding assays: ERLIN1/2 complexes interact selectively with monophosphorylated phosphoinositides, with strongest binding to PI(3)P (~5-fold greater than to PI(4)P and PI(5)P)

• Cholesterol measurement protocols: When assessing ERLIN1's role in cholesterol homeostasis, distinguish between total cellular cholesterol, membrane-bound cholesterol, and cholesterol esters

• ERAD pathway components: Monitor additional proteins including RNF170, TMUB1, and IP3Rs, as ERLIN1 functions within larger protein complexes

• Subcellular compartment markers: Include markers for ER, Golgi, and lipid droplets to accurately interpret ERLIN1's role in cholesterol transport between organelles

How can contradictory data about ERLIN1 expression patterns across different studies be reconciled?

Contradictory data regarding ERLIN1 expression patterns can be reconciled by considering several technical and biological factors:

• Antibody specificity: Many anti-ERLIN1 antibodies cross-react with unrelated proteins. For example, analysis showed that some antibodies produce nucleolar staining that cannot be suppressed by siRNA against TMUB1, indicating non-specific binding

• Complex formation dependencies: ERLIN1 stability depends on complex formation with ERLIN2. In ERLIN2 knockout cells, ERLIN1 may show altered expression patterns due to reduced stability rather than reflecting true tissue-specific expression

• Detection methods: Western blotting of membrane fractions versus whole-cell lysates can yield different results. Immunohistochemistry results depend heavily on fixation and antigen retrieval methods (TE buffer pH 9.0 versus citrate buffer pH 6.0)

• Functional redundancy: In some experimental systems, ERLIN1 and ERLIN2 show compensatory expression. A comprehensive approach using multiple antibodies and detection methods, along with genetic knockdown validation, is essential to resolve contradictory data

What is the optimal protein extraction protocol for detecting ERLIN1 in membrane fractions?

For optimal ERLIN1 detection in membrane fractions:

• Buffer composition: Use a buffer containing 20 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM EDTA, and protease inhibitor cocktail

• Membrane isolation:

  • Homogenize cells/tissues in buffer using Dounce homogenizer (20-25 strokes)

  • Centrifuge at 1,000 g for 10 minutes to remove nuclei and unbroken cells

  • Ultracentrifuge supernatant at 100,000 g for 1 hour at 4°C

  • Resuspend membrane pellet in buffer containing 1% Triton X-100

• Detergent resistance analysis:

  • For lipid raft studies, solubilize membranes in cold 1% Triton X-100

  • Layer on Optiprep gradient (5-40%)

  • Ultracentrifuge at 200,000 g for 4 hours

  • Collect fractions from top (DRMs float to lower density fractions)

• Storage:

  • Flash-freeze aliquots in liquid nitrogen

  • Store at -80°C; avoid repeated freeze-thaw cycles

The ratio of ERLIN2 to ERLIN1 in membrane extracts is typically ~2:1, which can serve as a quality control metric for membrane preparations .

How can immunoprecipitation of ERLIN1 be optimized to study its interaction partners?

Optimizing ERLIN1 immunoprecipitation requires specific considerations:

• Antibody selection: Use 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate; polyclonal antibodies targeting the C-terminal region of ERLIN1 typically perform better than those targeting the N-terminus

• Lysis conditions:

  • Mild lysis: 1% digitonin or 0.5% CHAPS preserves ERLIN1/2 complex integrity

  • Standard lysis: 1% Triton X-100 with 150mM NaCl maintains most interactions

  • Stringent lysis: 1% NP-40 with 300mM NaCl reduces non-specific binding

• Bead selection:

  • For detecting ubiquitinated species (e.g., IP3R1): use TUBEs (Tandem Ubiquitin Binding Entities) conjugated to agarose

  • For general IP: Protein A/G magnetic beads show less non-specific binding than sepharose

• Controls:

  • IgG control: Crucial as some antibodies cross-react with nucleolar proteins

  • Input: Load 5-10% of pre-IP lysate

  • ERLIN1 knockout cells: Essential negative control

• Elution strategy:

  • For mass spectrometry: On-bead tryptic digestion

  • For immunoblotting: Elution with 2X SDS sample buffer at 70°C (not 95°C) for 10 minutes preserves membrane protein integrity

What approaches can effectively distinguish between ERLIN1's direct binding partners versus indirect complex components?

To distinguish direct ERLIN1 binding partners from indirect associations:

• Crosslinking strategies:

  • Use membrane-permeable crosslinkers like DSP (dithiobis(succinimidyl propionate)) at 0.5-2 mM

  • For zero-length crosslinking, employ EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) which links only directly interacting proteins

  • Perform sequential immunoprecipitation followed by mass spectrometry

• Proximity labeling:

  • Generate ERLIN1-BioID or ERLIN1-APEX2 fusion proteins

  • Incubate cells with biotin (BioID) or biotin-phenol (APEX2)

  • Streptavidin pull-down followed by mass spectrometry identifies proteins in close proximity

• Reconstitution systems:

  • Express recombinant ERLIN1 in membrane mimetic systems (nanodiscs or liposomes)

  • Test direct binding using purified candidate interactors

  • Mutations in key domains (e.g., T65I analogous mutations) can validate binding interfaces

• Domain-specific analysis:

  • ERLIN1 and its partners (TMUB1-L, RNF170) contain conserved motifs in their luminal domains

  • Three-dimensional modeling shows these motifs bind the stomatin/prohibitin/flotillin/HflKC domain of adjacent ERLIN subunits at different interfaces

  • Point mutations in these interfaces can differentiate direct from indirect interactions

Research has demonstrated that the ERLIN scaffolds are required for TMUB1-RNF170 interaction, as knockout of ERLINs completely prevents this association while preserving TMUB1-RNF185-TMEM259 interactions .

What are the critical parameters for immunohistochemical detection of ERLIN1 in tissue samples?

For optimal immunohistochemical detection of ERLIN1:

For dual immunofluorescence with other ER markers, optimal antibody dilution may need further optimization to 1:20-1:200 .

How can researchers accurately quantify changes in ERLIN1 expression across different experimental conditions?

For accurate quantification of ERLIN1 expression changes:

• Western blot quantification:

  • Use membrane fractions rather than whole cell lysates

  • Include loading controls specific to membrane proteins (calnexin or Na⁺/K⁺-ATPase)

  • Employ a standard curve using recombinant ERLIN1 for absolute quantification

  • Note that ERLIN1 levels increase by ~70-95% in cells lacking ERLIN2, suggesting compensatory regulation

• qPCR analysis:

  • Design primers spanning exon-exon junctions

  • Validate with melt curve analysis to confirm specificity

  • Use multiple reference genes (GAPDH alone is insufficient)

  • Account for the possibility of altered mRNA stability versus protein stability

• Image analysis for IHC/IF:

  • Use automated threshold-based quantification

  • Analyze multiple fields (minimum 10) per sample

  • Implement blinded scoring by multiple observers

  • Consider subcellular localization patterns, not just intensity

• Statistical considerations:

  • Account for the 2:1 ratio of ERLIN2:ERLIN1 in interpreting relative changes

  • IP3R1 levels can increase by ~65-94% in the absence of ERLINs, serving as an internal verification of functional changes

What are the functional assays to validate ERLIN1 antibody specificity in cellular systems?

To validate ERLIN1 antibody specificity through functional assays:

• Genetic manipulation controls:

  • Compare staining patterns in wild-type versus ERLIN1 knockout cells

  • Use siRNA knockdown with multiple sequences targeting different regions

  • Rescue experiments with re-expression of ERLIN1 in knockout lines

  • The DKO+E1/2 cell model (ERLIN1/2 double knockout with re-expressed proteins) provides an excellent validation system

• Subcellular fractionation correlation:

  • Antibody signals should be enriched in ER membrane fractions

  • Signal should correlate with detergent-resistant membrane fractions in density gradients

• Functional readouts:

  • Binding partners: Loss of antibody signal should correlate with disrupted binding to known partners (RNF170, TMUB1)

  • IP3R1 ubiquitination: ERLIN1 knockout reduces IP3R1 ubiquitination by ~67-94%

  • Cholesterol homeostasis: ERLIN1/2 deficiency alters cholesterol esterification and ER-to-Golgi transport

• Mutant analysis:

  • The T65I mutation prevents ERLIN1/2 complex formation and IP3R1 binding

  • Antibodies should distinguish between functional and non-functional ERLIN1 variants in appropriate assays