BTK Antibodies

Tyrosine-protein kinase BTK (BTK)

Non-receptor tyrosine kinase indispensable for B lymphocyte development, differentiation and signaling. Binding of antigen to the B-cell antigen receptor (BCR) triggers signaling that ultimately leads to B-cell activation.

Following BCR engagement and activation at the plasma membrane, phosphorylates PLCG2 at many sites, sparking the downstream signaling pathway through calcium mobilization, followed by activation of the protein kinase C (PKC) family members. PLCG2 phosphorylation is done in close collaboration with the adapter protein B-cell linker protein BLNK.

Gene Information

Human
Gene Name:	BTK
Uniprot:	Q06187
Entrez:	695

Family

Belongs to:
protein kinase superfamily

Alternative Names

Agammaglobulinaemia tyrosine kinase; AGMX1; AGMX1MGC126262; AT; ATKIMD1; B-cell progenitor kinase; BPK; Bruton agammaglobulinemia tyrosine kinase; Bruton tyrosine kinase; BTK; dominant-negative kinase-deficient Brutons tyrosine kinase; EC 2.7.10; EC 2.7.10.2; IMD1; PSCTK1; tyrosine-protein kinase BTK; XLA; XLAMGC126261

Molecular Weight

Mass (kDA):
76.281 kDA

Genomic Context

Human
Location:	Xq22.1
Sequence:	X; NC_000023.11 (101349447..101390796, complement)

Tissue Specificity

Predominantly expressed in B-lymphocytes.

Subcellular Localizations

Cytoplasm. Cell membrane; Peripheral membrane protein. Nucleus. In steady state, BTK is predominantly cytosolic. Following B-cell receptor (BCR) engagement by antigen, translocates to the plasma membrane through its PH domain. Plasma membrane localization is a critical step in the activation of BTK. A fraction of BTK also shuttles between the nucleus and the cytoplasm, and nuclear export is mediated by the nuclear export receptor CRM1.

Diseases

• Agammaglobulinemia
• X-linked Agammaglobulinemia
• Immunologic Deficiency Syndromes
• Leukemia
• Infective Disorder
• Genetic Diseases, X-linked
• Lymphoma
• Malignant Neoplasms
• Primary Immune Deficiency Disorder
• Neoplasms
• Bacterial Infections
• B-cell Lymphomas
• Inflammation

• Arthritis
• Hypogammaglobulinemia
• Autoimmune Reaction
• Tissue Adhesions
• Ischemia
• Rheumatoid Arthritis
• Autoimmune Diseases

Interacting Proteins

• ARID3A
• BLNK
• GTF2I
• HSP90AB1
• MAL

• MEOX2
• PRKCQ
• SH3BP5
• WAS

Pathways

• Cell Development
• Secretion
• Cell Activation
• Cell Differentiation
• Localization
• Cytokine Production
• B Cell Differentiation
• Pathogenesis
• Cell Death
• Cell Proliferation
• Immune Response
• B Cell Activation
• B Cell Proliferation

• Cell Maturation
• Mast Cell Activation
• Cell Cycle
• Chemotaxis
• Cell Growth
• Platelet Aggregation
• Hypersensitivity

PTMs

• Phosphorylation
• Dephosphorylation
• Cleavage
• Methylation
• Acetylation

• Sumoylation
• Demethylation
• Biotinylation
• Ribosylation
• Ubiquitination

Research Areas

• Immunology

For details, visit:
bosterbio.com/bosterbio-gene-info-cards/BTK

Best Uses of the BTK Marker

This article outlines the most effective uses of BTK markers in various applications and diseases. The protein was purified from PBMCs, amplified PCR BTK residues were detected, and the phosphorylation status of BTK was quantified by Western blot. This article also explains the effectiveness of Brukinsa over imbruvica. Read on to learn more about the BTK marker.

PCR amplified BTK residues

Boster Bio carried out PCR amplification using a primer that was specific to the gene of concern. After TOPO was cloned into the pFastBac vector (KF), Life Technologies produced BTK residues 391-659 fusioned to a Histidine 6X tag. BTK expression was carried out in Sf9 cells for 48 hours. Following that, the cells were removed by centrifugation. The BTK pellets were stored at -80°C until further purification.

Poseltinib inhibits BTK the phosphorylation process in dose-dependent ways. Poseltinib slowed BTK phosphorylation and AKT phosphorylation in human PBMCs. The drug is able to treat many inflammatory diseases.

Recent studies confirm the role of Btk. Mice deficient in Btk have diminished capacity to form B cells and lack the most cytoplasmic portion of the B-cell receptor-associated signaling molecule Ig-a. XLA sufferers are affected by a mutation in the gene that results in deficiency in the protein. It is also known that Btk mutations in humans result in the X-linked immunodeficiency.

Western blot assay

The Boster Bio BTK marker is a widely used tool to detect protein expressing the cytokeratin B receptor. This antibody recognizes cells with the BTK gene. In addition to its role in the production of immunoglobulins, BTK plays a significant role in several immune cell types. It is crucial for the maturation and proliferation of B cells as well as their differentiation into Ab-producing plasma cells. It also plays a role in the regulation of cytokine, inflammatory mediators and platelet activation via glycoprotein VI receptors. It also regulates osteoclast differentiation in response RANK signaling and cell movement in response to chemokines.

For the Boster Bio BTK marker Western Blot assay, one x 106 cells were plated and stained for viability with violet-fixable dye. 0.02 Sodium Azide is contained in the FC staining buffer (4% FCS 1x PBS). The cells were then blocked with the appropriate mouse IgG solution (Microsoft, 1 mg/ml) for 1 hour at ambient temperature in the dark.

Optimized staining allowed for resolution of subpopulations within BM and naive cells as well as analysing phosphoproteins linked to the BCR. Phosphorylation of Syk was measured in B cells that were naive and um and also in Sm CD27+ and CD27 B cells, which are generally IgM-negative. Phosphorylation of Syk was not significantly different between the two cells.

Detection of phosphorylated BTK in PBMCs.

PBMCs were obtained from healthy individuals and incubated at 37°C for a day in 5% CO2. The PBMCs then were taken from healthy volunteers and incubated for 1 day at 37°C in 5 percent CO2. The specimens were labeled and stored in an ultra-low-temperature fridge. The samples were then processed for immunofluorescence staining. For the test one sample of PBMCs is compared to the same sample from the control group.

PBMCs were transfected with kB-luciferase (80 ng) and Renilla-luciferase (40 ng) Plasmids that express CD4-TLR8 and CD4-TLR9 as well as Btk(K430R) or phosphorylated BTK. Phosphorylated BTK in PBMCs was detected in these PBMCs by Western blot analysis.

It is important to identify which cells have BTK the phosphorylation process, since it is an important signaling molecule in the BCR Cytokine pathway. Phosphorylated BTK is found in the B lymphocytes' cytoplasm mast leukocytes, and bone marrow cells. It is involved in signaling pathways like cell proliferation, cytokine production and antigen presentation.

After establishing the cell-surface marker, PBMC were stained using anti-IgG antibodies and anti-IgA ones. This allowed pSyk detection in CD27+ cells and CD27 cells. However, naive B cell cultures did not express the pSyk gene. This immunofluorescence assay was validated by using FCB which allows for multiple time intervals in a single tube. The fluorescent-cell-barcoded samples were deconvoluted for further analysis.

The study was conducted using mice as a model for IgAN PK-PK, and healthy controls. The results revealed that BTK was expressed by patients suffering from the disease in their renal tissues. The levels of phosphatrylated BTK were significantly higher in IgAN kidney tissues than control kidney tissues. Both methods of immunostaining identified significant differences in BTK expression. These results confirmed the relationship of BTK with the presence of complement reactivity in kidneys.

After stimulation, immunocytochemistry was employed to determine the degree of phosphorylation. The samples were fixed in 4% paraformaldehyde, and incubated with primary antibodies against total BTK and monoclonal antibodies of rabbit anti-human against the latter. Secondary antibodies were used to identify the virus. ImageJ software was used to analyze the data. It is important to note that the response to BTK was variable.

Effectiveness of Brukinsa over Imbruvica

While the FDA has yet to approve either drug both companies have begun a head-to-head trial to test their efficacy in treating patients suffering from aggressive forms of the disease. Beijing-based biotech BeiGene began the trial in November. It was comprised of patients suffering from mantle cells lymphomawhich is an aggressive form of blood cancer. The drug has demonstrated promise, but has had a low success rate in clinical trials.

Beigene plans to share its findings with regulators in Europe and the United States, and present its data at medical conferences. It will continue to assess the effectiveness of Brukinsa in comparison to Imbruvica as part of its ongoing Phase III ALPINE trial. The study currently enrolls patients suffering from relapsed or refractory CLL.

While both drugs performed well in the initial trials but BeiGene was more successful. BeiGene drug has shown superior control of the progression of cancer in comparison to Imbruvica. Its safety profile was better in the study than Imbruvica as it had a pre-specified risk reduction of 60% in Brukinsa patients compared to 10.1% for Imbruvica. However, both drugs have some serious side effects, like atrial fibrillation. Patients who have irregular heartbeats are at higher risk of having a stroke or developing heart failure.

Brukinsa was approved last year, has not received the same approval. In a late-stage study it failed to meet the primary endpoint that demonstrated superiority in the complete response rate over Imbruvica. The company hopes to position its drug as the leading choice for this type of cancer treatment. The shares of the company jumped by 6 percent following the announcement of the trial results.