ube2z Antibody

What is UBE2Z and what is its role in the ubiquitination pathway?

UBE2Z, also known as Use1 (Uba6-specific E2 conjugating enzyme 1), is a 38-43 kDa member of the Ubiquitin-conjugating (E2) enzyme family that is highly expressed in human placenta, pancreas, spleen, and testis. It contains an E2 catalytic core domain with an active site cysteine that forms a thioester bond with ubiquitin . UBE2Z is unique because it functions specifically with the UBE1L2/UBA6 ubiquitin-activating (E1) enzyme, rather than the more common UBE1 E1 enzyme .

The protein mediates the ubiquitination of proteins like RGS4 and RGS5 via the N-end rule proteolytic pathway. Additionally, UBE2Z works with UBA6 to mediate the conjugation of FAT10, a ubiquitin-like protein, and can become auto-FAT10ylated in response to TNF-alpha and IFN-gamma . The full-length human UBE2Z is 354 amino acids and shares 100% sequence identity with mouse and rat UBE2Z .

UBE2Z is classified as a class IV E2 enzyme because it contains both N- and C-terminal extensions in addition to the core UBC domain, distinguishing it from the majority of E2 enzymes that contain only the core domain . These extensions likely play important roles in determining specificity and interaction with various E3 ligases.

What are the key applications for UBE2Z antibodies in research?

UBE2Z antibodies are valuable tools for investigating ubiquitination pathways, with several validated applications:

Western Blotting (WB): Most UBE2Z antibodies are validated for WB applications with recommended dilutions typically ranging from 1:500-1:2000 . Western blot is useful for detecting UBE2Z protein expression levels in cell lysates and tissue samples. The observed molecular weight is typically around 45 kDa despite calculated molecular weights of 28-38 kDa .

Immunohistochemistry (IHC): Several UBE2Z antibodies are suitable for IHC applications, with dilutions typically ranging from 1:50-1:500 . IHC allows visualization of UBE2Z distribution in tissue sections, with demonstrated success in human and mouse kidney, human testis, and other tissues .

Immunofluorescence (IF): UBE2Z antibodies can be used for immunofluorescence to study subcellular localization, with typical dilutions around 1:50-1:100 . Search results show successful IF in cell lines like MCF-7 and A-431 .

ELISA: Some UBE2Z antibodies are validated for ELISA applications, expanding the quantitative analysis options .

Validation of RNAi Experiments: UBE2Z antibodies are essential for confirming knockdown efficiency in RNAi experiments targeting UBE2Z .

Why might UBE2Z's molecular weight vary in Western blot results?

UBE2Z displays interesting variations in molecular weight across different experimental systems:

These variations can be attributed to several factors:

• Post-translational modifications: UBE2Z may undergo modifications like phosphorylation, ubiquitination, or FAT10ylation that increase its apparent molecular weight. As an E2 enzyme involved in FAT10 conjugation, UBE2Z can become auto-FAT10ylated in response to TNF-alpha and IFN-gamma .

• Protein isoforms: The existence of alternative splicing variants is suggested by references to "a potential smaller isoform lacks aa 1-108" .

• Species differences: Although human, mouse, and rat UBE2Z share high sequence identity, there might be slight variations in size or post-translational modifications across species.

• Technical factors: Differences in gel systems, running conditions, or sample preparation can affect the apparent molecular weight of proteins in Western blots.

When interpreting Western blot results for UBE2Z, researchers should consider these potential variations and include appropriate positive controls from tissues known to express high levels of UBE2Z, such as testis, placenta, or pancreas.

What protein domains should I target when selecting a UBE2Z antibody?

UBE2Z antibodies target various domains of the protein, each with specific advantages:

• N-Terminal region: Several antibodies target this region:

  • Antibodies targeting AA 9-35 of the N-terminal region

  • The sequence starting with "MAQAEGAYHR"

• Middle region: Some antibodies target the middle region (AA 136-225)

• C-Terminal region: Antibodies targeting the C-terminal portion can provide different specificity

• Full-length protein: Some antibodies are raised against the entire protein (AA 1-246)

When selecting a UBE2Z antibody, consider:

• The UBC core domain is highly conserved among E2 enzymes, so antibodies targeting this region might cross-react with other E2s

• UBE2Z's unique N- and C-terminal extensions make these regions potential targets for specific recognition

• For cross-species studies, target conserved regions, as UBE2Z shows high sequence identity between human, mouse, and rat

• Match the target region to your experimental needs - for example, if studying domain-specific functions or interactions

How can I validate the specificity of UBE2Z antibodies in my experimental system?

Thorough validation is essential to ensure antibody specificity:

• RNAi knockdown validation:

  • UBE2Z antibodies can be validated using target-specific siRNA, as shown in multiple search results

  • The antibody should detect a band in control siRNA-transfected cells that is significantly reduced in UBE2Z siRNA-transfected cells

• Overexpression validation:

  • Test cells transfected with UBE2Z expression vector versus empty vector control

  • Some antibodies are specifically noted as "reactive against mammalian transfected lysate"

• Tissue/cell type validation:

  • Test the antibody in tissues known to express high levels of UBE2Z (placenta, pancreas, spleen, and testis)

  • Multiple antibodies show successful detection in various human cell lines (MCF7, HepG2, HeLa, A549, PANC-1) and tissues (testis, kidney)

• Multiple antibodies approach:

  • Use multiple antibodies targeting different epitopes of UBE2Z and confirm consistent results

  • This helps rule out non-specific binding and confirms the identity of the detected protein

• Molecular weight verification:

  • Verify that the detected band is at the expected molecular weight (approximately 38-45 kDa)

  • Be aware of potential isoforms or post-translational modifications

What are the best controls to use when conducting UBE2Z knockdown experiments?

For reliable UBE2Z knockdown studies, incorporate these essential controls:

• Negative controls:

  • Non-targeting control siRNA/shRNA: As demonstrated in search result "U-138MG cells transfected with the control siRNA"

  • Untransfected cells: To establish baseline expression levels

• Multiple knockdown constructs:

  • Use at least two different siRNA/shRNA sequences targeting different regions of UBE2Z

  • This distinguishes between on-target and off-target effects

• Rescue controls:

  • Re-expression of siRNA/shRNA-resistant UBE2Z (with silent mutations in the targeting sequence)

  • This confirms phenotype specificity and rules out off-target effects

• Protein expression validation:

  • Western blot with validated UBE2Z antibody to confirm reduction in protein levels

  • Consider using antibodies targeting different epitopes for comprehensive detection

• Functional controls:

  • Assess levels of known UBE2Z substrates or interacting partners

  • Monitor ubiquitination levels of specific targets

  • For FAT10ylation studies, monitor FAT10 conjugation

• Time course analysis:

  • Assess knockdown efficiency at multiple time points to determine optimal experimental window

• Cell viability controls:

  • Since UBE2Z may be involved in apoptosis regulation , monitor cell viability

What is the optimal protocol for Western blot detection of UBE2Z?

Based on the search results, here's an optimized protocol for Western blot detection of UBE2Z:

Sample preparation:

• Human tissues: UBE2Z is detected in human testis tissue

• Mouse tissues: Successfully detected in mouse pancreas tissue and mouse kidney

• Cell lines: Detection in MCF7, HepG2, HeLa , A549, and PANC-1

Protocol steps:

• Lysate preparation:

  • Extract proteins using standard lysis buffers (RIPA or NP-40 based)

  • Include protease inhibitors (especially important for tissues with high protease activity like pancreas)

• SDS-PAGE:

  • Load 25μg of protein per lane

  • Use 4-12% gradient gels for optimal separation

• Transfer:

  • PVDF membrane is recommended

• Blocking:

  • 3% nonfat dry milk in TBST as blocking buffer

• Primary antibody:

  • Dilution range: 1:500-1:2000 for most UBE2Z antibodies

  • Alternatively, use 0.4-0.5 μg/mL for concentration-based dilutions

  • Incubate overnight at 4°C or 1-2 hours at room temperature

• Secondary antibody:

  • HRP-conjugated secondary antibody at 1:10000 dilution

  • Match to the primary antibody host species

• Detection:

  • ECL-based detection systems

  • Exposure time: Approximately 90 seconds, but optimize based on signal strength

• Expected results:

  • Look for a band at approximately 38-45 kDa

  • May vary slightly between species or due to post-translational modifications

How should I optimize IHC conditions for UBE2Z antibodies?

For optimal immunohistochemical detection of UBE2Z:

FFPE tissue optimization:

• Antibody selection:

  • Several antibodies are validated for IHC-P

• Dilution range:

  • 1:50 - 1:200 dilution is recommended

  • 1:200 dilution was successful for human testis tissue

• Tissue samples with validated results:

  • Human kidney, mouse kidney (1:100 dilution)

  • Human testis tissue

• Detection system:

  • Use an appropriate secondary antibody system compatible with your primary antibody host species

  • DAB (3,3'-diaminobenzidine) is commonly used as the chromogen

• Visualization:

  • 40x magnification is appropriate for detailed examination of subcellular localization

Immunofluorescence optimization:

• Fixation and permeabilization:

  • PFA fixation with Triton X-100 permeabilization has been verified for UBE2Z antibodies

• Antibody concentration:

  • 4μg/ml has been successfully used for IF

  • Alternative dilution recommendation: 1:50 - 1:100

• Expected staining pattern:

  • Predominantly cytoplasmic staining, as UBE2Z is "enriched in the cytoplasm"

  • Use DAPI for nuclear counterstaining

What are the differences between UBE2Z's role in ubiquitin versus FAT10 conjugation pathways?

UBE2Z is unusual in its ability to function in both ubiquitin and FAT10 conjugation pathways:

Ubiquitin pathway:

• Functions as an E2 enzyme downstream of UBA6 (E1) in ubiquitin conjugation to target proteins

• Specific to UBA6 and cannot be charged with ubiquitin by the more common UBE1 E1 enzyme

• Mediates ubiquitination of proteins like RGS4 and RGS5 via the N-end rule proteolytic pathway

• In the ubiquitination system, UBE2Z showed between 8-22 E3 interactions in a comprehensive framework of E2-RING E3 interactions

FAT10 pathway:

• Functions with UBA6 to mediate the conjugation of FAT10, a ubiquitin-like protein

• Specificity toward UBA6 and UBE2Z lies within the C-terminal CYCI tetrapeptide in FAT10

• This CYCI motif slows down transfer rates for FAT10 from UBA6 onto UBE2Z compared to ubiquitin

• UBE2Z can become auto-FAT10ylated in response to TNF-alpha and IFN-gamma

The crystal structure of UBE2Z provides functional insight into how specificity is achieved in these pathways. The structure was determined at 2.10Å resolution and reveals how the different domains of UBE2Z are organized :

This structural data combined with mutational analyses helps understand how UBE2Z achieves specificity in different conjugation pathways .

What are the recommended approaches for studying UBE2Z interactions with UBA6 and FAT10?

To study the interactions between UBE2Z, UBA6, and FAT10, consider these methodologies:

• In vitro transfer assays:

  • Reaction mix containing UBA6 (E1), ATP, UBL (ubiquitin or FAT10), and varying concentrations of UBE2Z (E2)

  • Stop reactions with non-reducing SDS-PAGE buffer (45s for reactions with ubiquitin, 4min for FAT10)

  • Separate by gel electrophoresis and quantify E2~UBL bands

  • Determine initial rates at different E2 concentrations and fit to Michaelis-Menten model

• Thioester bond verification:

  • Confirm the covalent nature of E2-UBL interaction via thioester bond by treating with reducing agents

  • The E2~UBL thioester bond should be reducible with 5mM β-mercaptoethanol

• Structural studies:

  • X-ray crystallography has been used to determine UBE2Z structure at 2.10Å resolution

  • This reveals domain organization relevant to UBA6 and FAT10 interactions

• Mutational analysis:

  • Study of the C-terminal CYCI tetrapeptide in FAT10 demonstrated its role in specificity

  • Similar approaches can identify key residues in UBE2Z that interact with UBA6 or FAT10

• Kinetic measurements:

  • Determine apparent Vmax and apparent Km values for the combined activity of the UBA6-UBE2Z couple

  • These measurements help understand the efficiency of the transfer reaction

• Stimulus-responsive studies:

  • UBE2Z is auto-FAT10ylated in response to TNF-alpha and IFN-gamma

  • These cytokines can be used to stimulate FAT10 conjugation pathways in cellular models

How can I determine if UBE2Z is involved in a specific protein's degradation pathway?

To investigate if your protein of interest (POI) is regulated by UBE2Z-mediated degradation:

• UBE2Z knockdown/knockout:

  • Use siRNA , shRNA, or CRISPR-Cas9 to reduce or eliminate UBE2Z expression

  • Monitor POI levels by Western blot to assess stability changes

  • Include proteasome inhibitors (e.g., MG132) to distinguish between synthesis and degradation effects

• UBE2Z overexpression:

  • Compare effects of wild-type UBE2Z versus catalytically inactive mutant (mutate active site cysteine)

  • Monitor changes in POI levels and ubiquitination status

• Protein half-life determination:

  • Perform cycloheximide chase assays in control versus UBE2Z-depleted cells

  • Measure POI decay rate to determine if UBE2Z affects its stability

• Direct ubiquitination assays:

  • Immunoprecipitate POI under denaturing conditions

  • Probe for ubiquitin to detect changes in ubiquitination upon UBE2Z manipulation

  • Consider using ubiquitin mutants (K48- or K63-only) to determine chain topology

• In vitro ubiquitination:

  • Set up reactions with purified components:

    • UBA6 (E1) - UBE2Z is specific for UBA6

    • UBE2Z (E2)

    • Candidate E3 ligases

    • POI as substrate

    • Ubiquitin, ATP, Mg²⁺

  • Detect ubiquitination by Western blot or mass spectrometry

• UBA6 dependency:

  • Confirm that UBA6 knockdown produces similar effects on POI as UBE2Z knockdown

  • This is expected since UBE2Z works specifically with UBA6

• FAT10 versus ubiquitin pathway:

  • Determine whether POI is modified by ubiquitin, FAT10, or both

  • Use FAT10 knockdown to distinguish between these pathways

How do I properly store and handle UBE2Z antibodies to maintain their activity?

Proper storage and handling are critical for maintaining antibody performance:

Storage temperature:

• Most UBE2Z antibodies should be stored at -20°C

• For R&D Systems antibody:

  • 12 months from date of receipt at -20 to -70°C as supplied

  • 1 month at 2 to 8°C under sterile conditions after reconstitution

  • 6 months at -20 to -70°C under sterile conditions after reconstitution

Storage buffer:

• Typically supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

Aliquoting recommendations:

• "Use a manual defrost freezer and avoid repeated freeze-thaw cycles"

• For some formulations, "Aliquoting is unnecessary for -20°C storage"

• For 20μl sizes, note that they may contain 0.1% BSA

Shipping and handling:

• Some antibodies are shipped on wet ice

• Upon receipt, store according to manufacturer recommendations

• Always centrifuge briefly before opening to ensure all material is at the bottom of the vial

Quality control considerations:

• Note the expiration date and lot number

• Test each new lot against a known positive control

• For critical applications, consider testing multiple antibody lots in parallel