FKBP19 Antibody

What is FKBP11 and what is its biological significance?

FKBP11 (FK506 Binding Protein 11, 19 kDa) belongs to the broader FKBP family of proteins that bind to the immunosuppressant Tacrolimus (FK-506) and possess peptidyl-prolyl cis-trans isomerase activity. FKBP11 plays a critical role in antibody folding processes and demonstrates plasma cell-specific expression patterns. Research indicates that FKBP11 is directly involved in catalyzing antibody folding, making it particularly significant in plasma cell biology and antibody production .

Unlike other FKBP family members, FKBP11 shows highly specific localization to antibody-producing plasma cells and is induced during B cell to plasma cell differentiation. This specific expression pattern suggests a specialized role in immunoglobulin production and processing within the adaptive immune system .

What antibody formats are available for FKBP11 detection?

Several antibody formats targeting different epitopes of FKBP11 are available for research applications:

Researchers should select the appropriate antibody based on their specific experimental needs, including the target epitope, required applications, and species reactivity .

How do FKBP11 antibodies differ from other FKBP family antibodies?

FKBP11 antibodies recognize a distinct member of the FKBP family that has unique functional and expression characteristics. While all FKBP family proteins share the ability to bind FK506 and exhibit peptidyl-prolyl isomerase activity, they differ in their tissue distribution, subcellular localization, and specialized functions.

The FKBP family in humans comprises multiple members including: AIP, AIPL1, FKBP1A, FKBP1B, FKBP2, FKBP3, FKBP5, FKBP6, FKBP7, FKBP8, FKBP9, FKBP9L, FKBP10, FKBP11, FKBP14, FKBP15, and FKBP52 . Unlike many other family members, FKBP11 demonstrates plasma cell-specific expression and specialized roles in antibody folding, which makes antibodies against FKBP11 particularly valuable for plasma cell research and investigations into antibody production mechanisms .

How can FKBP11 antibodies be used to study plasma cell development?

FKBP11 antibodies serve as excellent tools for tracking plasma cell differentiation due to the specific upregulation of FKBP11 during B cell to plasma cell transition. Research demonstrates that in vitro B cell to plasma cell differentiation is accompanied by significant induction of FKBP11 expression .

Methodologically, researchers can implement FKBP11 antibodies in flow cytometry protocols alongside established plasma cell markers such as CD38 and CD27. For comprehensive plasma cell phenotyping, consider the following multi-color flow cytometry panel:

• Anti-human CD3-Alexa Fluor 700 (T cell exclusion)

• Anti-human CD19-APC/Fire 750 (B cell lineage)

• Anti-human CD27-Brilliant Violet 605 (memory B cell/plasma cell)

• Anti-human CD38-eFluor 450 (plasma cell marker)

• Anti-human FKBP11 antibody (plasma cell-specific protein)

• FcR blocking reagent (to prevent non-specific binding)

• TO-PRO-3 (viability dye)

This panel allows for precise identification of plasma cells and assessment of FKBP11 expression levels during different stages of plasma cell development and in response to various stimuli.

What is the role of FKBP11 in antibody folding and how can this be experimentally assessed?

FKBP11 functions as a peptidyl-prolyl cis-trans isomerase specifically involved in antibody folding processes. Studies have demonstrated that recombinant human FKBP11 can efficiently refold IgG antibodies in vitro, and this activity is inhibited by the immunosuppressant FK506 .

Researchers can experimentally assess FKBP11's role in antibody folding using the following protocol:

• Denature a monoclonal antibody using 3 M guanidinium chloride (pH 7.0, 0.1 M Tris, 0.005 M EDTA) for 24 hours at 4°C.

• Initiate refolding by diluting the denatured antibody solution 10-fold in PBS containing recombinant FKBP11 (approximately 45 μM).

• At specific time points, withdraw aliquots and dilute 12-fold in trypsin solution to stop further refolding.

• Quantify correctly refolded antibody using ELISA.

• For inhibition studies, preincubate FKBP11 with FK506 (180 μM) or DMSO (control) for one hour before conducting the refolding assay .

This experimental approach allows for direct assessment of FKBP11's catalytic activity in antibody folding and evaluation of potential inhibitors or enhancers of this process.

What are the optimal conditions for using FKBP11 antibodies in Western blotting?

For optimal Western blotting results with FKBP11 antibodies, researchers should follow these methodological guidelines:

• Sample preparation: Extract proteins from cells or tissues using a lysis buffer containing protease inhibitors. For plasma cell-specific analysis, consider isolating CD38+CD27+ cells first.

• Gel electrophoresis: Resolve 20-30 μg of total protein on a 12-15% SDS-PAGE gel, as FKBP11 is a relatively small protein (19 kDa).

• Transfer: Use a PVDF membrane and transfer at 100V for 1 hour or 30V overnight at 4°C.

• Blocking: Block with 5% non-fat dry milk in TBST for 1 hour at room temperature.

• Primary antibody: Dilute anti-FKBP11 antibody according to manufacturer's recommendations (typically 1:1000 to 1:2000) in blocking buffer and incubate overnight at 4°C.

• Secondary antibody: After washing, apply appropriate HRP-conjugated secondary antibody (typically 1:5000) for 1 hour at room temperature.

• Detection: Develop using enhanced chemiluminescence and visualize using a digital imaging system .

Expected results include a distinct band at approximately 19 kDa corresponding to FKBP11, with potentially stronger signals in plasma cell-enriched samples compared to other B cell populations or non-lymphoid tissues.

How should immunofluorescence experiments be designed when using FKBP11 antibodies?

For successful immunofluorescence using FKBP11 antibodies, implement the following protocol for both cultured cells and tissue sections:

For cultured cells (IF-cc):

• Fix cells with 4% paraformaldehyde for 15 minutes at room temperature.

• Permeabilize with 0.2% Triton X-100 in PBS for 10 minutes.

• Block with 5% normal serum from the species of the secondary antibody for 1 hour.

• Incubate with anti-FKBP11 antibody (typically 1:100 to 1:500 dilution) overnight at 4°C.

• After washing, apply fluorophore-conjugated secondary antibody for 1 hour at room temperature.

• Counterstain nuclei with DAPI and mount with anti-fade mounting medium.

For paraffin-embedded tissue sections (IF-p):

• Deparaffinize and rehydrate sections using standard protocols.

• Perform antigen retrieval using citrate buffer (pH 6.0) for 20 minutes.

• Block endogenous peroxidase activity if applicable.

• Follow steps 3-6 as described for cultured cells .

For co-localization studies in plasma cells, consider pairing FKBP11 staining with markers such as CD138 or intracellular immunoglobulin chains to confirm plasma cell-specific expression and subcellular localization within the endoplasmic reticulum.

How can researchers address non-specific binding in FKBP11 antibody applications?

Non-specific binding is a common challenge when working with antibodies. For FKBP11 antibody applications, implement these strategies to minimize non-specific signals:

If non-specific binding persists, evaluate alternative antibody clones targeting different epitopes of FKBP11.

How do researchers interpret contradictory results between FKBP11 expression and function?

When faced with contradictory results regarding FKBP11 expression and function, consider these analytical approaches:

• Validate antibody specificity: Confirm the specificity of your FKBP11 antibody using positive controls (plasma cells) and negative controls (non-plasma cells). Consider using multiple antibodies targeting different epitopes of FKBP11.

• Assess inhibition controls: When studying enzymatic functions, include FK506 inhibition controls to confirm that the observed activity is specifically due to FKBP11's peptidyl-prolyl isomerase activity .

• Evaluate context-dependent expression: FKBP11 expression is highly plasma cell-specific, but might be induced in other cell types under ER stress conditions. Characterize the cellular context thoroughly, including differentiation state and stress conditions.

• Consider post-translational modifications: Investigate whether post-translational modifications affect FKBP11 function without altering detection by antibodies.

• Examine subcellular localization: Contradictory results may stem from differences in subcellular localization. Use fractionation approaches or high-resolution microscopy to determine precise localization patterns.

• Employ molecular approaches: Complement antibody-based studies with molecular techniques such as RT-PCR, RNA-seq, or CRISPR-based gene editing to correlate protein expression with mRNA levels and functional effects.

By systematically analyzing these factors, researchers can resolve apparent contradictions and develop a more nuanced understanding of FKBP11 biology in different experimental contexts.