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Monoclonal Antibody Immunoprecipitation Mitotic Cell Cycle

$122
20 µl
$293
100 µl
APPLICATIONS
REACTIVITY
Human, Rat

Application Methods: Flow Cytometry, Immunofluorescence (Immunocytochemistry), Immunoprecipitation, Western Blotting

Background: Cyclins are a family of proteins that activate specific cyclin-dependent kinases required for progression through the cell cycle. The entry of all eukaryotic cells into mitosis is regulated by activation of cdc2/cdk1 at the G2/M transition. This activation is a multi-step process that begins with the binding of the regulatory subunit, cyclin B1, to cdc2/cdk1 to form the mitosis-promoting factor (MPF). MPF remains in the inactive state until phosphorylation of cdc2/cdk1 at Thr161 by cdk activating kinase (CAK) (1,2) and dephosphorylation of cdc2/cdk1 at Thr14/Tyr15 by cdc25C (3-5). Five cyclin B1 phosphorylation sites (Ser116, 126, 128, 133, and 147) are located in the cytoplasmic retention signal (CRS) domain and are thought to regulate the translocation of cyclin B1 to the nucleus at the G2/M checkpoint, promoting nuclear accumulation and initiation of mitosis (6-9). While MPF itself can phosphorylate Ser126 and Ser128, polo-like kinase 1 (PLK1) phosphorylates cyclin B1 preferentially at Ser133 and possibly at Ser147 (6,10). At the end of mitosis, cyclin B1 is targeted for degradation by the anaphase-promoting complex (APC), allowing for cell cycle progression (11). Research studies have shown that cyclin B1 is overexpressed in breast, prostate, and non-small cell lung cancers (12-14).

$260
100 µl
APPLICATIONS
REACTIVITY
Human, Mouse, Rat

Application Methods: Immunoprecipitation, Western Blotting

Background: Correct segregation of sister chromatids prior to the onset of cell division is essential to the maintenance of genetic integrity and the avoidance of aneuploidy and chromosomal instability, characteristics of many cancer cells. The mitotic checkpoint, also known as the spindle assembly checkpoint, monitors accurate attachment of kinetochores to the spindle, inhibits mitosis and delays the onset of anaphase until all chromosomes are aligned at the metaphase plate (1). MAD2L1 is an essential participant in the mitotic checkpoint (2). It exists in two conformations, including the open and inactive O-MAD2 form and the closed, active C-MAD2 form. Prior to mitosis, MAD2L1 is localized to the cytosol and exists largely in the closed, inactive form. During the mitotic checkpoint, MAD2L1 switches to the open, active conformation (3). Together with other checkpoint proteins, MAD2L1 binds to and deactivates Cdc20, thereby inhibiting the anaphase promoting complex (4). When the kinetochores are correctly attached to the spindle, MAD2L1 releases Cdc20, which allows activation of the anaphase promoting complex and subsequent degradation of key mitotic substrates and the initiation of metaphase-anaphase transition (5).

$122
20 µl
$303
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey, Mouse, Rat

Application Methods: Immunoprecipitation, Western Blotting

Background: The retinoblastoma tumor suppressor protein Rb regulates cell proliferation by controlling progression through the restriction point within the G1-phase of the cell cycle (1). Rb has three functionally distinct binding domains and interacts with critical regulatory proteins including the E2F family of transcription factors, c-Abl tyrosine kinase, and proteins with a conserved LXCXE motif (2-4). Cell cycle-dependent phosphorylation by a CDK inhibits Rb target binding and allows cell cycle progression (5). Rb inactivation and subsequent cell cycle progression likely requires an initial phosphorylation by cyclin D-CDK4/6 followed by cyclin E-CDK2 phosphorylation (6). Specificity of different CDK/cyclin complexes has been observed in vitro (6-8) and cyclin D1 is required for Ser780 phosphorylation in vivo (9).

$111
20 µl
$260
100 µl
$630
300 µl
APPLICATIONS
REACTIVITY
Bovine, Human, Monkey, Pig

Application Methods: Chromatin IP, Flow Cytometry, Immunofluorescence (Immunocytochemistry), Immunohistochemistry (Paraffin), Immunoprecipitation, Western Blotting

Background: The retinoblastoma tumor suppressor protein Rb regulates cell proliferation by controlling progression through the restriction point within the G1-phase of the cell cycle (1). Rb has three functionally distinct binding domains and interacts with critical regulatory proteins including the E2F family of transcription factors, c-Abl tyrosine kinase, and proteins with a conserved LXCXE motif (2-4). Cell cycle-dependent phosphorylation by a CDK inhibits Rb target binding and allows cell cycle progression (5). Rb inactivation and subsequent cell cycle progression likely requires an initial phosphorylation by cyclin D-CDK4/6 followed by cyclin E-CDK2 phosphorylation (6). Specificity of different CDK/cyclin complexes has been observed in vitro (6-8) and cyclin D1 is required for Ser780 phosphorylation in vivo (9).

$260
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey, Mouse, Rat

Application Methods: Immunoprecipitation, Western Blotting

Background: Eukaryotic cell proliferation depends strictly upon the E3 ubiquitin ligase activity of the anaphase promoting complex/cyclosome (APC/C), whose main function is to trigger the transition of the cell cycle from metaphase to anaphase. The APC/C complex promotes the assembly of polyubiquitin chains on substrate proteins in order to target these proteins for degradation by the 26S proteasome (1,2). The vertebrate APC/C complex consists of as many as 15 subunits, including multiple scaffold proteins, two catalytic subunits (APC2, APC11), and a number of proteins responsible for substrate recognition (3). All E3 enzymes, including APC/C, utilize ubiquitin residues activated by E1 enzymes and transferred to E2 enzymes. Research studies indicate that APC/C interacts with the E2 enzymes UBE2S and UBE2C via the RING-finger domain-containing subunit APC11 (4-6). APC/C function relies on multiple cofactors, including an APC/C coactivator formed by the cell division control protein 20 homolog (CDC20) and Cdh1/FZR1. The CDC20/Cdh1 coactivator is responsible for recognition of APC/C substrates through interaction with specific D-box and KEN-box recognition elements within these substrates (7-9).

$260
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey, Mouse, Rat

Application Methods: Immunoprecipitation, Western Blotting

Background: The 20S proteasome is the major proteolytic enzyme complex involved in intracellular protein degradation. PA700, PA28, and PA200 are three major protein complexes that function as activators of the 20S proteasome. There are three evolutionarily conserved subunits of PA28: PA28α (PSME1), PA28β (PSME2), and PA28γ (PSME3) (1,2). PA28α and PA28β form a heteroheptameric complex and function by binding to the 20S complex at its opening site(s). The PA28α/β complex is present throughout the cell and participates in MHC class I antigen presentation by promoting the generation of antigenic peptides from foreign proteins (2). PA28γ exists in the form of a homoheptamer and is mainly located in the nucleus. The PA28γ complex exerts its function by binding and guiding specific nuclear target proteins to the 20S proteasome for further degradation (3,4).

$303
100 µl
APPLICATIONS
REACTIVITY
Human

Application Methods: Immunoprecipitation, Western Blotting

Background: Cyclins are a family of proteins that activate specific cyclin-dependent kinases required for progression through the cell cycle. The entry of all eukaryotic cells into mitosis is regulated by activation of cdc2/cdk1 at the G2/M transition. This activation is a multi-step process that begins with the binding of the regulatory subunit, cyclin B1, to cdc2/cdk1 to form the mitosis-promoting factor (MPF). MPF remains in the inactive state until phosphorylation of cdc2/cdk1 at Thr161 by cdk activating kinase (CAK) (1,2) and dephosphorylation of cdc2/cdk1 at Thr14/Tyr15 by cdc25C (3-5). Five cyclin B1 phosphorylation sites (Ser116, 126, 128, 133, and 147) are located in the cytoplasmic retention signal (CRS) domain and are thought to regulate the translocation of cyclin B1 to the nucleus at the G2/M checkpoint, promoting nuclear accumulation and initiation of mitosis (6-9). While MPF itself can phosphorylate Ser126 and Ser128, polo-like kinase 1 (PLK1) phosphorylates cyclin B1 preferentially at Ser133 and possibly at Ser147 (6,10). At the end of mitosis, cyclin B1 is targeted for degradation by the anaphase-promoting complex (APC), allowing for cell cycle progression (11). Research studies have shown that cyclin B1 is overexpressed in breast, prostate, and non-small cell lung cancers (12-14).

$260
100 µl
APPLICATIONS
REACTIVITY
Human

Application Methods: Immunoprecipitation, Western Blotting

Background: Emerin is a broadly expressed integral protein of the nuclear inner membrane (1). It contains a LEM domain and binds to several nuclear proteins, such as BAF (barrier-to-autointegration factor) and A- and B-type lamins, which are important in nuclear functions (2-5). Emerin may regulate gene expression through binding to other transcriptional regulators (6,7). Emerin binds to β-catenin and inhibits its nuclear accumulation (8). Recent studies demonstrate that emerin is required for HIV-1 infectivity (9). Mutations in the gene encoding emerin (EMD) are a major cause of Emery-Dreifuss muscular dystrophy (EDMD), a disorder characterized by progressive skeletal muscle weakening (10).

$303
100 µl
APPLICATIONS
REACTIVITY
Human

Application Methods: Immunoprecipitation, Western Blotting

Background: At least four distinct polo-like kinases exist in mammalian cells: PLK1, PLK2, PLK3, and PLK4/SAK (1). PLK1 apparently plays many roles during mitosis, particularly in regulating mitotic entry and exit. The mitosis promoting factor (MPF), cdc2/cyclin B1, is activated by dephosphorylation of cdc2 (Thr14/Tyr15) by cdc25C. PLK1 phosphorylates cdc25C at Ser198 and cyclin B1 at Ser133 causing translocation of these proteins from the cytoplasm to the nucleus (2-5). PLK1 phosphorylation of Myt1 at Ser426 and Thr495 has been proposed to inactivate Myt1, one of the kinases known to phosphorylate cdc2 at Thr14/Tyr15 (6). Polo-like kinases also phosphorylate the cohesin subunit SCC1, causing cohesin displacement from chromosome arms that allow for proper cohesin localization to centromeres (7). Mitotic exit requires activation of the anaphase promoting complex (APC) (8), a ubiquitin ligase responsible for removal of cohesin at centromeres, and degradation of securin, cyclin A, cyclin B1, Aurora A, and cdc20 (9). PLK1 phosphorylation of the APC subunits Apc1, cdc16, and cdc27 has been demonstrated in vitro and has been proposed as a mechanism by which mitotic exit is regulated (10,11).Substitution of Thr210 with Asp has been reported to elevate PLK1 kinase activity and delay/arrest cells in mitosis, while a Ser137Asp substitution leads to S-phase arrest (12). In addition, while DNA damage has been found to inhibit PLK1 kinase activity, the Thr210Asp mutant is resistant to this inhibition (13). PLK1 has been reported to be phosphorylated in vivo at Ser137 and Thr210 in mitosis; DNA damage prevents phosphorylation at these sites (14).

$260
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey, Mouse, Rat

Application Methods: Immunoprecipitation, Western Blotting

Background: Eukaryotic cell proliferation depends strictly upon the E3 ubiquitin ligase activity of the anaphase promoting complex/cyclosome (APC/C), whose main function is to trigger the transition of the cell cycle from metaphase to anaphase. The APC/C complex promotes the assembly of polyubiquitin chains on substrate proteins in order to target these proteins for degradation by the 26S proteasome (1,2). The vertebrate APC/C complex consists of as many as 15 subunits, including multiple scaffold proteins, two catalytic subunits (APC2, APC11), and a number of proteins responsible for substrate recognition (3). All E3 enzymes, including APC/C, utilize ubiquitin residues activated by E1 enzymes and transferred to E2 enzymes. Research studies indicate that APC/C interacts with the E2 enzymes UBE2S and UBE2C via the RING-finger domain-containing subunit APC11 (4-6). APC/C function relies on multiple cofactors, including an APC/C coactivator formed by the cell division control protein 20 homolog (CDC20) and Cdh1/FZR1. The CDC20/Cdh1 coactivator is responsible for recognition of APC/C substrates through interaction with specific D-box and KEN-box recognition elements within these substrates (7-9).

$260
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey

Application Methods: Immunoprecipitation, Western Blotting

Background: Eukaryotic cell proliferation depends strictly upon the E3 ubiquitin ligase activity of the anaphase promoting complex/cyclosome (APC/C), whose main function is to trigger the transition of the cell cycle from metaphase to anaphase. The APC/C complex promotes the assembly of polyubiquitin chains on substrate proteins in order to target these proteins for degradation by the 26S proteasome (1,2). The vertebrate APC/C complex consists of as many as 15 subunits, including multiple scaffold proteins, two catalytic subunits (APC2, APC11), and a number of proteins responsible for substrate recognition (3). All E3 enzymes, including APC/C, utilize ubiquitin residues activated by E1 enzymes and transferred to E2 enzymes. Research studies indicate that APC/C interacts with the E2 enzymes UBE2S and UBE2C via the RING-finger domain-containing subunit APC11 (4-6). APC/C function relies on multiple cofactors, including an APC/C coactivator formed by the cell division control protein 20 homolog (CDC20) and Cdh1/FZR1. The CDC20/Cdh1 coactivator is responsible for recognition of APC/C substrates through interaction with specific D-box and KEN-box recognition elements within these substrates (7-9).

$260
100 µl
APPLICATIONS
REACTIVITY
Hamster, Human, Monkey, Mouse, Rat

Application Methods: Immunoprecipitation, Western Blotting

Background: The entry of eukaryotic cells into mitosis is regulated by cdc2 kinase activation, a process controlled at several steps including cyclin binding and phosphorylation of cdc2 at Thr161 (1). However, the critical regulatory step in activating cdc2 during progression into mitosis appears to be dephosphorylation of cdc2 at Thr14 and Tyr15 (2). Phosphorylation at Thr14 and Tyr15, resulting in inhibition of cdc2, can be carried out by Wee1 and Myt1 protein kinases (3,4). The cdc25 phosphatase may be responsible for removal of phosphates at Thr14 and Tyr15 and subsequent activation of cdc2 (1,5).

$260
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey, Mouse, Rat

Application Methods: Immunoprecipitation, Western Blotting

Background: S5a (PSMD4) is a subunit of the 19S regulatory proteasome complex functioning in ubiquitinated-protein targeting and degradation (1). S5a contains two polyubiquitin binding motifs (UIM) that bind multiubiquitin chains by hydrophobic interaction (2,3). In addition to ubiquitin, the UIM of S5a shows high affinity to a ubiquitin-like domain present in many proteins. S5a binds to these types of proteins directly and mediates their targeting to the proteasome for degradation (4,5).

$260
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey, Mouse, Rat

Application Methods: Immunoprecipitation, Western Blotting

Background: The 26S proteasome is a highly abundant proteolytic complex involved in the degradation of ubiquitinated substrate proteins. It consists largely of two sub-complexes, the 20S catalytic core particle (CP) and the 19S/PA700 regulatory particle (RP) that can cap either end of the CP. The CP consists of two stacked heteroheptameric β-rings (β1-7) that contain three catalytic β-subunits and are flanked on either side by two heteroheptameric α-rings (α1-7). The RP includes a base and a lid, each having multiple subunits. The base, in part, is composed of a heterohexameric ring of ATPase subunits belonging to the AAA (ATPases Associated with diverse cellular Activities) family. The ATPase subunits function to unfold the substrate and open the gate formed by the α-subunits, thus exposing the unfolded substrate to the catalytic β-subunits. The lid consists of ubiquitin receptors and DUBs that function in recruitment of ubiquitinated substrates and modification of ubiquitin chain topology (1,2). Other modulators of proteasome activity, such as PA28/11S REG, can also bind to the end of the 20S CP and activate it (1,2).

$260
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey, Mouse, Rat

Application Methods: Immunoprecipitation, Western Blotting

Background: Structural maintenance of chromosomes 2 (SMC2) and 4 (SMC4) proteins are subunits of the condensin complex, which enables chromosome condensation and maintains the compaction of chromosomes as they separate to opposite poles during anaphase (1-3). In addition to regulating chromosome condensation, condensin is a general regulator of chromosome architecture and may function to regulate gene expression and DNA repair. SMC proteins contain a hallmark bipartite ATPase domain of the ABC ATPase superfamily, which consists of an N-terminal Walker A motif nucleotide-binding domain and C-terminal Walker B motif catalytic domain that interact to form a functional ATPase (1-3). The two ATPase domains are connected by two coiled coil domains separated by a central hinge region that facilitates protein-protein interactions between partnering SMC proteins. In the case of the condensin complex, SMC2 and SMC4 interact to form a functional ATPase required for chromatin condensation; however, the mechanism by which this ATPase activity regulates chromsome architecture is still being determined. In addition to SMC proteins, condensin contains three auxiliary subunits, which function to regulate condensin ATPase activity. Higher eukaryotes contain two distinct condensin complexes (condensin I and II), both of which contain SMC2 and SMC4 (1-3). Condensin I also contains the auxiliary subunits CAP-D2, CAP-G and CAP-H, while condensin II contains the related auxiliary proteins CAP-D3, CAP-G2 and CAP-H2. The two condensin complexes show different localization patterns during the cell cycle and on chromosomes and both are required for successful mitosis, suggesting distinct functions for each complex (1-3).

$134
20 µl
$336
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey, Mouse, Rat

Application Methods: Flow Cytometry, Immunofluorescence (Immunocytochemistry), Immunohistochemistry (Paraffin), Immunoprecipitation, Western Blotting

Background: The retinoblastoma tumor suppressor protein Rb regulates cell proliferation by controlling progression through the restriction point within the G1-phase of the cell cycle (1). Rb has three functionally distinct binding domains and interacts with critical regulatory proteins including the E2F family of transcription factors, c-Abl tyrosine kinase, and proteins with a conserved LXCXE motif (2-4). Cell cycle-dependent phosphorylation by a CDK inhibits Rb target binding and allows cell cycle progression (5). Rb inactivation and subsequent cell cycle progression likely requires an initial phosphorylation by cyclin D-CDK4/6 followed by cyclin E-CDK2 phosphorylation (6). Specificity of different CDK/cyclin complexes has been observed in vitro (6-8) and cyclin D1 is required for Ser780 phosphorylation in vivo (9).

$111
20 µl
$260
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey, Mouse

Application Methods: Chromatin IP, Immunoprecipitation, Western Blotting

Background: The retinoblastoma tumor suppressor protein Rb regulates cell proliferation by controlling progression through the restriction point within the G1-phase of the cell cycle (1). Rb has three functionally distinct binding domains and interacts with critical regulatory proteins including the E2F family of transcription factors, c-Abl tyrosine kinase, and proteins with a conserved LXCXE motif (2-4). Cell cycle-dependent phosphorylation by a CDK inhibits Rb target binding and allows cell cycle progression (5). Rb inactivation and subsequent cell cycle progression likely requires an initial phosphorylation by cyclin D-CDK4/6 followed by cyclin E-CDK2 phosphorylation (6). Specificity of different CDK/cyclin complexes has been observed in vitro (6-8) and cyclin D1 is required for Ser780 phosphorylation in vivo (9).

$260
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey, Mouse, Rat

Application Methods: Immunoprecipitation, Western Blotting

Background: S5a (PSMD4) is a subunit of the 19S regulatory proteasome complex functioning in ubiquitinated-protein targeting and degradation (1). S5a contains two polyubiquitin binding motifs (UIM) that bind multiubiquitin chains by hydrophobic interaction (2,3). In addition to ubiquitin, the UIM of S5a shows high affinity to a ubiquitin-like domain present in many proteins. S5a binds to these types of proteins directly and mediates their targeting to the proteasome for degradation (4,5).

$260
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey, Mouse, Rat

Application Methods: Immunoprecipitation, Western Blotting

Background: The cell division cycle demands accuracy to avoid the accumulation of genetic damage. This process is controlled by molecular circuits called "checkpoints" that are common to all eukaryotic cells (1). Checkpoints monitor DNA integrity and cell growth prior to replication and division at the G1/S and G2/M transitions, respectively. The cdc2-cyclin B kinase is pivotal in regulating the G2/M transition (2,3). Cdc2 is phosphorylated at Thr14 and Tyr15 during G2-phase by the kinases Wee1 and Myt1, rendering it inactive. The tumor suppressor protein retinoblastoma (Rb) controls progression through the late G1 restriction point (R) and is a major regulator of the G1/S transition (4). During early and mid G1-phase, Rb binds to and represses the transcription factor E2F (5). The phosphorylation of Rb late in G1-phase by CDKs induces Rb to dissociate from E2F, permitting the transcription of S-phase-promoting genes. In vitro, Rb can be phosphorylated at multiple sites by cdc2, cdk2, and cdk4/6 (6-8). DNA damage triggers both the G2/M and the G1/S checkpoints. DNA damage activates the DNA-PK/ATM/ATR kinases, which phosphorylate Chk at Ser345 (9), Chk2 at Thr68 (10) and p53 (11). The Chk kinases inactivate cdc25 via phosphorylation at Ser216, blocking the activation of cdc2.

$303
100 µl
APPLICATIONS
REACTIVITY
Human

Application Methods: Immunoprecipitation, Western Blotting

Background: Ubiquitin is a conserved polypeptide unit that plays an important role in the ubiquitin-proteasome pathway. Ubiquitin can be covalently linked to many cellular proteins by the ubiquitination process, which targets proteins for degradation by the 26S proteasome. Three components are involved in the target protein-ubiquitin conjugation process. Ubiquitin is first activated by forming a thiolester complex with the activation component E1; the activated ubiquitin is subsequently transferred to the ubiquitin-carrier protein E2, then from E2 to ubiquitin ligase E3 for final delivery to the epsilon-NH2 of the target protein lysine residue (1-3). The ubiquitin-proteasome pathway has been implicated in a wide range of normal biological processes and in disease-related abnormalities. Several proteins such as IκB, p53, cdc25A, and Bcl-2 have been shown to be targets for the ubiquitin-proteasome process as part of regulation of cell cycle progression, differentiation, cell stress response, and apoptosis (4-7).