USP36 Antibody, Biotin conjugated

Basic Research Questions

• What is USP36 and what are its primary functions in cellular processes?

  USP36, also known as ubiquitin specific peptidase 36, is a deubiquitinating enzyme (DUB) belonging to the Peptidase C19 protein family. The canonical human USP36 protein consists of 1123 amino acid residues with a molecular mass of approximately 122.9 kDa .

  USP36 has multiple critical cellular functions:

  • Regulates ribosome biogenesis through deubiquitination of nucleolar proteins

  • Controls chromatin organization through histone modification

  • Participates in autophagy pathways

  • Regulates neuronal signaling through TrkA receptor deubiquitination

  • Promotes SUMOylation of small nucleolar ribonucleoprotein (snoRNP) components

  • Inhibits apoptosis by deubiquitinating cIAP1 and survivin in certain cancers

  Methodologically, researchers studying USP36 function should consider cell-type specific expression patterns, as USP36 is widely expressed across many tissues but may have tissue-specific roles.

• How is USP36 localized in cells and why is this significant for experimental design?

  USP36 exhibits dynamic subcellular localization that researchers should consider when designing experiments. While earlier studies using HeLa cells indicated primarily nuclear (particularly nucleolar) localization, more recent research demonstrates:

  • In PC12-6/15 cells, USP36 localizes to nucleoli but also accumulates in non-nuclear compartments

  • Fractionation experiments reveal that USP36 primarily localizes to non-nuclear compartments in some cell types

  • USP36 can partially co-localize with TrkA receptors, suggesting functional interactions at the membrane/cytoplasmic interface

  When designing immunofluorescence experiments, researchers should:

  • Include appropriate subcellular markers (nucleolar, cytoplasmic, and membrane)

  • Perform fractionation experiments to confirm localization patterns in their specific cell type

  • Consider using confocal microscopy for co-localization studies with potential interaction partners

• What applications are supported for USP36 antibody, biotin conjugated?

  Based on product information and literature, USP36 antibody (biotin conjugated) supports several research applications:

  Application	Validated	Recommended Dilution	Notes
  ELISA	Yes	Optimal dilutions to be determined by end user	Primary validated application
  Western Blot	Yes	1:500-1:1000	Detected in Jurkat, HeLa, and HepG2 cells
  Immunoprecipitation	Yes	0.5-4.0 μg for 1.0-3.0 mg total protein	Validated in Jurkat cells
  Immunofluorescence	Yes	1:200-1:800	Validated in U2OS cells
  Co-IP	Yes	See specific protocols	Useful for interaction studies

  For optimal results, researchers should:

  • Titrate the antibody for each specific application and cell type

  • Include appropriate positive and negative controls

  • Validate antibody specificity using knockdown or knockout approaches

• What is the significance of biotin conjugation in USP36 antibody applications?

  Biotin conjugation provides several methodological advantages that researchers can leverage:

  • Enhanced detection sensitivity through the strong biotin-streptavidin interaction system

  • Flexibility in detection methods (can be used with various streptavidin-conjugated reporters)

  • Compatibility with multi-color immunofluorescence studies

  • Reduced background in tissues with high endogenous immunoglobulin levels

  • Compatibility with tyramide signal amplification (TSA) for ultra-sensitive detection

  When using biotin-conjugated USP36 antibody, researchers should:

  • Block endogenous biotin when working with biotin-rich tissues

  • Consider using streptavidin-conjugated fluorophores or enzymes appropriate for their detection system

  • Be aware that the biotin conjugation might affect the antibody's binding characteristics compared to unconjugated versions

• What are the optimal storage and handling conditions for biotin-conjugated USP36 antibody?

  Based on manufacturer specifications, researchers should follow these guidelines:

  • Store at -20°C for long-term storage

  • Some manufacturers recommend -80°C storage as an alternative

  • Avoid repeated freeze/thaw cycles by making small aliquots

  • Store in buffer containing 50% glycerol, 0.01M PBS, pH 7.4, with 0.03% Proclin-300 as preservative

  • Stable for one year after shipment when properly stored

  For optimal antibody performance:

  • Bring to room temperature before opening

  • Centrifuge briefly before use

  • Return to appropriate storage temperature immediately after use

Advanced Research Questions

• How can USP36 antibody be used to investigate neuronal signaling through TrkA regulation?

  USP36 has been shown to interact with TrkA and regulate its activation kinetics . To investigate this relationship:

  1. Experimental approach for co-localization studies:

     • Perform double immunofluorescence using biotin-conjugated USP36 antibody and TrkA antibody

     • Use high-resolution confocal microscopy to visualize potential co-localization

     • Apply stimulation with nerve growth factor (NGF) to assess dynamic changes

  2. Interaction studies methodology:

     • Utilize biotin-conjugated USP36 antibody for pull-down assays with streptavidin beads

     • Perform co-immunoprecipitation experiments to confirm endogenous interaction

     • Use proximity ligation assay (PLA) for in situ visualization of protein interactions

  3. Functional analysis:

     • Combine USP36 knockdown with TrkA ubiquitination assays

     • Use biotin-conjugated USP36 antibody to monitor USP36 expression levels

     • Measure TrkA activation kinetics (phosphorylation) following NGF stimulation in USP36-depleted cells

  The research by Iribarren et al. demonstrated that "USP36 knockdown affects TrkA activation kinetics and USP36 interacts with TrkA," suggesting direct regulation .

• What experimental designs can elucidate USP36's role in SUMOylation processes?

  USP36 has been shown to promote SUMOylation of snoRNP components Nop58 and Nhp2 . To investigate this function:

  1. In vitro SUMOylation assays:

     • Reconstitute SUMOylation reactions using recombinant E1 (SAE1/SAE2), E2 (Ubc9), SUMO2, ATP, and purified substrate

     • Add recombinant USP36 to assess enhancement of SUMOylation

     • Use biotin-conjugated USP36 antibody to immunodeplete USP36 from cell extracts and test effect on SUMOylation capacity

  2. Interaction studies:

     • Use GST pull-down assays to confirm direct binding between USP36 and components of SUMOylation machinery

     • Perform domain mapping to identify regions of USP36 responsible for SUMO and Ubc9 binding

     • Delineate the N-terminal region (aa 1-420) of USP36 that binds preferentially to polymeric SUMO2 chains

  3. Cellular SUMOylation assays:

     • Transfect cells with His-tagged SUMO constructs

     • Perform Ni-NTA pulldowns under denaturing conditions

     • Use biotin-conjugated USP36 antibody to confirm USP36 expression levels

     • Analyze SUMOylation of specific substrates like snoRNP components

  Research has shown that "USP36 drastically increased PARP1 SUMOylation in vitro," demonstrating its SUMOylation-promoting activity .

• How can researchers validate USP36 antibody specificity in their experimental systems?

  Validation of antibody specificity is critical for reliable results. For USP36 antibody:

  1. Genetic validation approaches:

     • Use CRISPR/Cas9 to generate USP36 knockout cell lines

     • Employ siRNA or shRNA to knockdown USP36 expression

     • Verify disappearance or reduction of signal in Western blot, immunofluorescence, or ELISA assays

  2. Expression validation:

     • Overexpress tagged USP36 constructs (GFP-USP36 or FLAG-USP36)

     • Perform parallel detection with tag-specific antibodies and USP36 antibody

     • Confirm signal co-localization and intensity correlation

  3. Biochemical validation:

     • Pre-absorb antibody with immunizing peptide (recombinant USP36 fragment 670-881 AA)

     • Perform peptide competition assays to confirm signal specificity

     • Test antibody against multiple cell lines with known USP36 expression levels (Jurkat, HeLa, HepG2)

  4. Cross-reactivity assessment:

     • Test in cells from different species (human antibody shows less cross-reactivity with mouse samples)

     • Evaluate potential cross-reactivity with other USP family members

     • Consider comparing signal pattern with multiple different USP36 antibodies

• What protocols are recommended for using biotin-conjugated USP36 antibody in co-immunoprecipitation studies?

  For effective co-immunoprecipitation studies with biotin-conjugated USP36 antibody:

  1. Standard Co-IP protocol:

     • Prepare cell lysates in non-denaturing buffer (150 mM NaCl, 50 mM Tris pH 7.5, 1% NP-40)

     • Pre-clear lysates with protein A/G beads to reduce non-specific binding

     • Incubate with biotin-conjugated USP36 antibody (2-5 μg per mg of protein)

     • Capture complexes using streptavidin-coated magnetic beads

     • Wash extensively (4-5 times) with IP buffer containing reduced detergent

     • Elute by boiling in SDS-PAGE sample buffer

  2. Modified protocol for detecting transient interactions:

     • Include crosslinking step with DSP (dithiobis[succinimidyl propionate])

     • Use proteasome inhibitors (MG132) to stabilize ubiquitinated complexes

     • Add deubiquitinase inhibitors (PR-619, NEM) to preserve ubiquitin modifications

  3. For studying specific interactions:

     • For TrkA interaction: stimulate cells with NGF before lysis

     • For Nedd4-2 interaction: co-transfect cells with GFP-Nedd4-2 constructs

     • For snoRNP components: perform nuclear fractionation before IP

  Research has shown successful co-immunoprecipitation of USP36 with TrkA: "TrkA and USP36 were pulled down after USP36 or TrkA immunoprecipitation, respectively" .

• How can USP36 antibody be used to investigate interactions with E3 ubiquitin ligases?

  USP36 has been shown to interact with the E3 ubiquitin ligase Nedd4-2 . To investigate such interactions:

  1. Mapping interaction domains:

     • Use GST-fusion proteins containing specific domains of Nedd4-2 (such as WW domains)

     • Perform pull-down assays with lysates from cells expressing USP36

     • Use biotin-conjugated USP36 antibody to detect bound USP36

     • Research has shown "USP36 interacted with WW domains 1, 3, and 4" of Nedd4-2

  2. Competition assays:

     • Perform pull-down assays with increasing amounts of USP36 to assess disruption of TrkA binding to Nedd4-2

     • Quantify binding through Western blot analysis

     • The study showed "increasing amounts of USP36 disrupted the binding of TrkA to the GST-WW3 protein"

  3. Functional impact assessment:

     • Analyze ubiquitination status of shared substrates (like TrkA) in the presence/absence of USP36

     • Investigate whether USP36 affects the activity or stability of the E3 ligase

     • Research showed "USP36 prevents TrkA ubiquitination mediated by Nedd4-2" through competitive binding

  4. Reciprocal regulation analysis:

     • Examine whether E3 ligases like Nedd4-2 can ubiquitinate USP36

     • Test if USP36 can deubiquitinate the E3 ligase

     • Study found "Nedd4-2 protein seems to be less ubiquitinated in the presence of exogenous USP36"

• What experimental designs are optimal for studying USP36's role in cancer-related pathways?

  USP36 inhibits apoptosis by deubiquitinating cIAP1 and survivin in cancer . For cancer-related studies:

  1. Cell survival and apoptosis assays:

     • Use biotin-conjugated USP36 antibody to monitor expression levels in different cancer cell lines

     • Perform USP36 knockdown followed by apoptosis assays (Annexin V/PI, caspase activity)

     • Assess impact on chemotherapy resistance in cancer cell models

  2. Substrate-specific deubiquitination analysis:

     • Implement in vitro deubiquitination assays with purified components

     • Use linkage-specific ubiquitin antibodies to determine chain types (K11-linked for cIAP1, K48-linked for survivin)

     • Perform cycloheximide chase assays to assess protein stability following USP36 manipulation

  3. Xenograft tumor models:

     • Establish stable USP36 knockdown or overexpression cancer cell lines

     • Monitor tumor growth rates in vivo

     • Use biotin-conjugated USP36 antibody for immunohistochemistry on tumor sections

     • Correlate USP36 levels with apoptotic markers and patient outcomes

  4. Mechanistic studies:

     • Investigate how "USP36 binds and removes lysine-11–linked ubiquitin chains from cIAP1 and lysine-48–linked ubiquitin chains from survivin to abolish protein degradation"

     • Perform structure-function studies to identify domains required for substrate recognition

• What are the recommended protocols for USP36 antibody in RNA processing and ribosome biogenesis studies?

  USP36 plays an important role in RNA processing and ribosome biogenesis:

  1. Nucleolar function analysis:

     • Use biotin-conjugated USP36 antibody for immunofluorescence co-staining with nucleolar markers

     • Perform nucleolar isolation followed by Western blot analysis

     • Detect USP36 in nucleolar fractions using biotin-conjugated antibody

  2. RNA helicase interaction studies:

     • Co-immunoprecipitate USP36 with DHX33 RNA helicase

     • Research has shown that "ectopically expressed GFP–USP36 could be detected in FLAG-tagged DHX33 immunoprecipitates"

     • Analyze DHX33 stability in USP36-depleted cells

  3. snoRNP SUMOylation analysis:

     • Implement SUMO-specific pull-down assays

     • Detect SUMOylated forms of Nop58, Nop56, Nhp2, and DKC1

     • Correlate with USP36 expression levels

     • Research showed "USP36 associates with both box C/D and Box H/ACA snoRNPs and promotes SUMOylation of two proteins in each complex"

  4. rRNA processing analysis:

     • Pulse-chase labeling of rRNA with 5-FU

     • Northern blot analysis of rRNA processing intermediates

     • Correlate processing defects with USP36 expression levels

     • Use biotin-conjugated USP36 antibody to confirm knockdown efficiency