ULBP1 Antibody, HRP conjugated

What is ULBP1 and what role does it play in immune function?

ULBP1 (UL16-binding protein 1) is a cell-surface protein that functions as a ligand for human NKG2D. It binds and activates the KLRK1/NKG2D receptor, thereby mediating natural killer cell cytotoxicity . ULBP1 belongs to a family of proteins that are upregulated under various stress conditions including viral infection, heat shock, and tumor transformation . It is distantly related to MHC class I proteins but possesses only the alpha 1 and alpha 2 Ig-like domains, without the capacity to bind peptide or interact with beta 2-microglobulin . ULBP1 is anchored to the cell membrane via a GPI-linkage, which has implications for its mobility within the membrane and its detection in experimental systems .

What are the alternative names for ULBP1 in the literature?

ULBP1 appears under several alternative nomenclatures in scientific literature, which is important to recognize when conducting literature searches. These include: N2DL1, RAET1I (retinoic acid early transcript 1I), UL16-binding protein 1, ALCAN-beta, NKG2D ligand 1, N2DL-1, and NKG2DL1 . The name ULBP derives from the original identification of these proteins as ligands for the human cytomegalovirus glycoprotein UL16, hence "UL16 binding proteins" .

What are the recommended applications for anti-ULBP1 antibodies?

Anti-ULBP1 antibodies have been validated for several experimental applications. The rabbit recombinant monoclonal ULBP1 antibody (such as Abcam's EPR7532(2) clone, ab176566) is suitable for Western blot (WB) and intracellular flow cytometry applications . When selecting an anti-ULBP1 antibody, researchers should ensure it has been validated for their specific application and species of interest. For flow cytometry applications, fluorophore-conjugated antibodies like PerCP-conjugated anti-ULBP1 (e.g., R&D Systems FAB1380C) can be particularly useful for multi-parameter analysis .

What are the optimal Western blot conditions for ULBP1 detection?

For Western blot applications, anti-ULBP1 antibodies typically perform optimally at a 1/1000 dilution . The Western blot protocol should include appropriate secondary antibodies, such as goat anti-rabbit HRP at 1/2000 dilution, and visualization using the ECL technique . For cell lysate preparation, several methods have been documented, including digitonin lysis buffer (1% digitonin, 150 mM NaCl, 50 mM Tris-HCl [pH 7.4], with protease inhibitors) or 1% NP-40 lysis buffer with protease inhibitors . To ensure equivalent loading across samples, normalization to total protein concentration (determined by BCA assay) and inclusion of loading controls such as anti-tubulin antibodies is recommended .

How should flow cytometry experiments with ULBP1 antibodies be designed?

For flow cytometry applications, researchers should consider both the fluorophore selection and appropriate controls. The antibody concentration should be optimized, with reported working dilutions ranging from 1/100 for intracellular staining to manufacturer-recommended concentrations for surface staining. Essential controls include isotype-matched control antibodies, fluorescence minus one (FMO) controls, and both positive and negative cell lines to establish gating strategies . For intracellular flow cytometry, proper permeabilization protocols must be followed. Gate setting should utilize unstained controls, single-stained controls, and secondary antibody alone controls to ensure accurate population identification .

How does viral infection affect ULBP1 expression and detection?

Viruses have evolved mechanisms to evade immune surveillance by modulating ULBP1 expression or detection. For example, HHV-6B infection results in downregulation of several NKG2D ligands including ULBP1 . The HHV-6B U20 glycoprotein binds directly to ULBP1 with sub-micromolar affinity, which decreases NKG2D binding to ULBP1 at the cell surface . Importantly, this mechanism does not decrease ULBP1 protein levels either at the cell surface or in total, but rather masks ULBP1 from detection by NKG2D . Researchers studying ULBP1 in the context of viral infections should therefore consider both protein expression levels and functional accessibility to receptor binding.

What strategies can be used to create stable ULBP1-expressing cell lines for antibody validation?

Creating stable ULBP1-expressing cell lines is a valuable approach for antibody validation and functional studies. This can be achieved through lentiviral transduction of the full coding sequence of ULBP1 (Uniprot Q9BZM6) into appropriate host cells . Selection markers such as puromycin resistance or IRES-GFP reporters can facilitate the isolation of transduced cells . To ensure consistent ULBP1 expression, cells should be sorted by anti-ULBP1 staining using flow cytometry . Different cell lines may be appropriate depending on the experimental question, with HEK293S GnTI cells suitable for studies requiring modified glycan structures and HEK293T for experiments needing fully intact glycans .

How can researchers investigate ULBP1 protein-protein interactions?

For investigating ULBP1 interactions with binding partners, several approaches are available. Co-immunoprecipitation can be performed using digitonin lysis buffer (1% digitonin, 150 mM NaCl, 50 mM Tris-HCl [pH 7.4]) supplemented with protease inhibitors to preserve protein-protein interactions . The choice of detergent is critical, as digitonin can help solubilize lipid rafts while maintaining protein complexes . Pull-down assays can be performed using antibodies against ULBP1 (such as Abcam ab176566) or epitope tags if working with tagged constructs . Visualization of interacting proteins can be achieved through Western blotting with appropriate antibodies and detection methods like enhanced chemiluminescence (ECL) .

How should researchers validate the specificity of their ULBP1 antibodies?

Antibody specificity must be rigorously validated to ensure reliable experimental results. Multiple approaches should be employed, including:

• Negative controls such as isotype-matched control antibodies

• Testing against known positive and negative cell lines

• Verification using lysates from cells transfected with ULBP1 compared to mock-transfected controls

• Confirmation of expected molecular weight (28 kDa) in Western blot applications

• For HRP-conjugated antibodies, testing for non-specific binding or high background by performing control experiments without primary antibody

What factors might affect the detection of ULBP1 by antibodies in experimental systems?

Several factors can influence ULBP1 detection by antibodies:

• Post-translational modifications: ULBP1 undergoes glycosylation, which may affect antibody binding. Different cell lines (e.g., HEK293S GnTI vs. HEK293T) can be used to study glycosylation effects .

• Protein masking: Viral proteins like HHV-6B U20 can bind to ULBP1 and mask antibody epitopes without reducing protein levels .

• Cell fixation and permeabilization: For intracellular staining, the fixation and permeabilization method can affect epitope accessibility.

• Buffer composition: Lysis buffers containing appropriate detergents (e.g., digitonin, NP-40) and protease inhibitors are critical for maintaining protein integrity during extraction .

• Protein trafficking: ULBP1 can be redirected to lysosomes under certain conditions, which may necessitate treatment with lysosomal inhibitors like leupeptin and folimycin to stabilize the protein .

How can researchers overcome issues with non-specific binding of HRP-conjugated antibodies?

Non-specific binding is a common challenge with HRP-conjugated antibodies. To mitigate this issue:

• Optimize blocking conditions using different blocking agents (BSA, milk, commercial blocking buffers)

• Increase washing stringency and duration

• Titrate the antibody concentration to determine the optimal signal-to-noise ratio

• Pre-absorb the antibody with proteins from the species being studied

• Consider using more specific detection methods like enhanced chemiluminescence (ECL)

• Employ proper negative controls including isotype controls and secondary-only controls

• When possible, validate findings using alternative detection methods or differently conjugated antibodies

How is ULBP1 detection relevant to cancer research?

ULBP1 plays a significant role in tumor immunosurveillance through its interaction with the NKG2D receptor on NK cells. Accurate detection of ULBP1 is therefore crucial for cancer research applications, including:

• Assessing NK cell activation potential against tumor cells

• Evaluating tumor immune evasion mechanisms

• Developing immunotherapeutic approaches targeting the NKG2D pathway

• Investigating the prognostic value of ULBP1 expression in different cancer types

• Understanding how cancer cells may downregulate or shed ULBP1 to evade immune detection

Research has demonstrated ULBP1 expression in various cancer cell lines including Jurkat (human acute T cell leukemia) and Molt-4 (human myeloid leukemia) , making these valuable positive controls for antibody validation.

What methodological considerations are important when studying ULBP1 in viral infection models?

When investigating ULBP1 in viral infection models, researchers should consider:

• The distinction between protein expression level and functional accessibility: viruses like HHV-6B can mask ULBP1 without decreasing its expression

• Time course experiments to capture dynamic changes in ULBP1 expression during infection

• Comparative analysis between infected and uninfected cells to identify virus-specific effects

• Use of viral mutants lacking specific immune evasion genes to delineate mechanisms

• Complementary approaches to detect ULBP1, including flow cytometry for surface expression and Western blotting for total protein levels

• Functional assays to assess NK cell activation in response to infected cells, which provides a readout of biologically relevant ULBP1 accessibility