Review

multichannel pipette (CELLTREAT Scientific)

Name: multichannel pipette
Brand: CELLTREAT Scientific
Rating: 92 (14 reviews)

CELLTREAT Scientific is a verified supplier

CELLTREAT Scientific manufactures this product

About

News

Press Release

Team

Advisors

Partners

Contact

Bioz Stars

Bioz vStars

Buy from Supplier

Structured Review

CELLTREAT Scientific multichannel pipette
Multichannel Pipette, supplied by CELLTREAT Scientific, used in various techniques. Bioz Stars score: 92/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/multichannel pipette/product/CELLTREAT Scientific
Average 92 stars, based on 14 article reviews

multichannel pipette - by Bioz Stars, 2026-03

92/100 stars

Images

Image Search Results

Domain structure, regulatory motifs, and interaction sites between RAS/RAF/KSR/MEK/14-3-3. (A) The domain structures of human KRAS, BRAF, KSR1, and MEK1 are shown and numbered according to the listed Uniprot accession numbers. Domain boundaries are marked based on structural data available in the PDB or are based on structure-based sequence alignments. 14-3-3 binding sites are based on published studies (Müller, Ory, Copeland, Piwnica-Worms, & Morrison, 2001; Xing, Kornfeld, & Muslin, 1997); in BRAF, these binding sites have been recently confirmed via structural studies (Kondo et al., 2019; Liau, Wendorff, et al., 2020; Park et al., 2019). In KSR1, the 14-3-3 binding sites are based on experimental data or predicted based on structural alignments (Cacace et al., 1999). (B) A cartoon depiction of the putative signalsome including KSR1 highlighting expected interactions sites based on known structures of fragments, as well as functional data (Brennan et al., 2011; Dhawan, Scopton, & Dar, 2016; Khan et al., 2020; Kondo et al., 2019; Lavoie et al., 2018; Liau, Wendorff, et al., 2020; Park et al., 2019).

Journal: Methods in enzymology

Article Title: Conformational control and regulation of the pseudokinase KSR via small molecule binding interactions

doi: 10.1016/bs.mie.2022.03.039

Figure Lengend Snippet: Domain structure, regulatory motifs, and interaction sites between RAS/RAF/KSR/MEK/14-3-3. (A) The domain structures of human KRAS, BRAF, KSR1, and MEK1 are shown and numbered according to the listed Uniprot accession numbers. Domain boundaries are marked based on structural data available in the PDB or are based on structure-based sequence alignments. 14-3-3 binding sites are based on published studies (Müller, Ory, Copeland, Piwnica-Worms, & Morrison, 2001; Xing, Kornfeld, & Muslin, 1997); in BRAF, these binding sites have been recently confirmed via structural studies (Kondo et al., 2019; Liau, Wendorff, et al., 2020; Park et al., 2019). In KSR1, the 14-3-3 binding sites are based on experimental data or predicted based on structural alignments (Cacace et al., 1999). (B) A cartoon depiction of the putative signalsome including KSR1 highlighting expected interactions sites based on known structures of fragments, as well as functional data (Brennan et al., 2011; Dhawan, Scopton, & Dar, 2016; Khan et al., 2020; Kondo et al., 2019; Lavoie et al., 2018; Liau, Wendorff, et al., 2020; Park et al., 2019).

Article Snippet: HEK293T cells (ATCC CRL-3216) Opti-MEM without Phenol Red (Gibco, Cat# 11058021) DMEM (Gibco, Cat# 11965092) PBS (Gibco, Cat# 20012027) 25 mL reservoirs (ThermoFisher, Cat# 95128093) Trypsin-EDTA (0.25%) with phenol red (Gibco, Cat# 25200114) White 96-well plates (Corning, Cat# 3990) Clear plastic 96-well plate (Celltreat, Cat# 229196)) Serological pipette tips (2mL aspirating, 2, 5, 10mL) Fugene HD Transfection Reagent (Promega, Cat# 2311) Carrier DNA (Promega, Cat# E4882) MEK inhibitors (Selleckchem) DMSO (Sigma, Cat# 275855-100mL) Countess slides (ThermoFisher, Cat# C10228) Promega NL substate and Inhibitor kit (Promega, Cat# N2161) Tracer Dilution Buffer (Promega, Cat# N2191) Tram-bo ( Khan et al., 2020 ) CMV-mKSR1, CMV-mKSR1-NL, and CMV-MEK1-NL DNA ( Khan et al., 2020 ) Stackable pipette tips (Rainin, Cat# 17005873 and 17005875, respectively) PCR tube strips (ThermoFisher, Cat# AB2000) 1.5 mL microcentrifuge tubes (ThermoFisher, Cat# 05-4080130) Trypan Blue (ThermoFisher, Cat# C10282) Tissue culture dishes (Celltreat, Cat# 229660) 15 mL Falcon tubes (Falcon, Cat# 352196)

Techniques: Sequencing, Binding Assay, Functional Assay

Structural features of KSR:MEK and RAF–MEK complexes. Tetrameric assembly of KSR1:MEK (A) and BRAF:MEK (B). The available crystal structures of KSRI-bound MEK (PDB ID: 7JV1; Khan et al., 2020) provide a snap-shot of KSR:KSR homodimerization centered around R665. MEK binds KSR on the flanking sides of the KSR homodimer. The left inset represents the heterodimerization interface between KSR1 and MEK1 centered around the respective helix ⊠G (W831 of KSR1 is shown as spheres) and the activation loops, which form a 2-stranded antiparallel β-sheet. The right inset highlights the location of the KSR1 orthosteric pocket with AMP-PNP bound and interfacial glue pocket. (B) A crystal structure of BRAF:MEK (PDB ID: 4MNE; Haling et al., 2014) highlighting BRAF homodimerization centered around R509. The lower insets highlight the homodimerization interface of KSR1 and BRAF. Whereas KSR1 homodimerization involves an extensive interaction of residues at the N-lobe of the two KSR1 molecules that results in a back-back dimer, two molecules of BRAF are assembled in an inverted fashion from N- to C-lobe. (C) Orthosteric pockets (left;bound to ADP, PDB ID 7JUQ; Khan et al., 2020) and interfacial binding pockets (middle left;bound to APS-2-79, PDB ID 5KKR; Dhawan et al., 2016). In KSR2, APS-2-79 directly projects toward a back pocket that is lined by hydrophobic residues Y714 (helix ⊠C), F725 (strand β4) and F825 (DFG motif). The binding of APS-2-79 results in the restructuring of the activation loop, downstream of the DFG motif (not shown) and disordering of P-loop (Dhawan et al., 2016). Right two panels show interfacial binding pockets of KSR1-MEK (PDB ID 7JV1) and KSR2-MEK (PDB ID 7JUV) occupied with trametiglue. The terminal sulfamide motif of trametiglue occupies the interfacial space between KSR and MEK, with complementarity between trametiglue and KSR spanning the pre-helix ⊠G loop and the tip of the helix ⊠G (Khan et al., 2020).

Journal: Methods in enzymology

Article Title: Conformational control and regulation of the pseudokinase KSR via small molecule binding interactions

doi: 10.1016/bs.mie.2022.03.039

Figure Lengend Snippet: Structural features of KSR:MEK and RAF–MEK complexes. Tetrameric assembly of KSR1:MEK (A) and BRAF:MEK (B). The available crystal structures of KSRI-bound MEK (PDB ID: 7JV1; Khan et al., 2020) provide a snap-shot of KSR:KSR homodimerization centered around R665. MEK binds KSR on the flanking sides of the KSR homodimer. The left inset represents the heterodimerization interface between KSR1 and MEK1 centered around the respective helix ⊠G (W831 of KSR1 is shown as spheres) and the activation loops, which form a 2-stranded antiparallel β-sheet. The right inset highlights the location of the KSR1 orthosteric pocket with AMP-PNP bound and interfacial glue pocket. (B) A crystal structure of BRAF:MEK (PDB ID: 4MNE; Haling et al., 2014) highlighting BRAF homodimerization centered around R509. The lower insets highlight the homodimerization interface of KSR1 and BRAF. Whereas KSR1 homodimerization involves an extensive interaction of residues at the N-lobe of the two KSR1 molecules that results in a back-back dimer, two molecules of BRAF are assembled in an inverted fashion from N- to C-lobe. (C) Orthosteric pockets (left;bound to ADP, PDB ID 7JUQ; Khan et al., 2020) and interfacial binding pockets (middle left;bound to APS-2-79, PDB ID 5KKR; Dhawan et al., 2016). In KSR2, APS-2-79 directly projects toward a back pocket that is lined by hydrophobic residues Y714 (helix ⊠C), F725 (strand β4) and F825 (DFG motif). The binding of APS-2-79 results in the restructuring of the activation loop, downstream of the DFG motif (not shown) and disordering of P-loop (Dhawan et al., 2016). Right two panels show interfacial binding pockets of KSR1-MEK (PDB ID 7JV1) and KSR2-MEK (PDB ID 7JUV) occupied with trametiglue. The terminal sulfamide motif of trametiglue occupies the interfacial space between KSR and MEK, with complementarity between trametiglue and KSR spanning the pre-helix ⊠G loop and the tip of the helix ⊠G (Khan et al., 2020).

Techniques: Activation Assay, Binding Assay

$Purification of the KSR1:MEK1 complex for biochemical and structural analysis. (A) Copurification of the KSR1 pseudokinase domain and MEK1 kinase domain is shown as an example (Khan et al., 2020). KSR1 [residues 591–899] and MEK1 [residues 33–393] were coexpressed as His-tagged proteins using the Sf21 insect cell system. (B) Cobaltaffinity purification of KSR1-MEK1 complex using the gravity-flow column (left);the corresponding 8–16% SDS-gel is shown on the right. (C) Partially-purified KSR1:MEK1 complex is further purified using anion exchange chromatography to remove excessive MEK. Several impurities are detected on the 8–16% SDS gel (below). (D) KSR1-MEK1 (1:1) complex is obtained after size-exclusion chromatography. Either the KSR1:MEK1 complex can be isolated as His-tagged protein (left) or KSR1-MEK1 can be treated with trypsin (1:1000, incubated overnight at 4 °C) to remove partially unfolded proteins and flexible loops, and hence highly homogenous proteins are obtained for crystallization. SDS gels are shown below, and the protein fractions corresponding to the tetrameric peak (150 kDa—see Fig. 2 for a tetrameric assembly) are pooled, and concentrated for subsequent analysis. (E) The flow-chart is provided to illustrate the procedure for determining structures of the KSR1:MEK1 complexes bound to interfacial binders, including trametinib (Khan et al., 2020). Similar procedures can be used for solving structures of other MEKi. Cocrystallization of the KSR:MEK1 complex in the presence of orthosteric binders can also be used to solve structures (Brennan et al., 2011; Dhawan, Scopton, & Dar, 2016). (left to right) Crystals of purified KSR1:MEK1 or KSR2:MEK1 complexes are first grown in the presence of 5mM AMP-PNP, and typically first appear within 24 h at room temperature. Crystals of KSR2-MEK1 are shown as an example. Next, individual crystals (72–96 h old) are transferred into a fresh drop containing crystallization solution (20% PEG2000, 200mM Mg acetate, 100mM MES pH 6.5) supplemented with 5 mM AMP-PNP and 1 mM trametinib. The drop is re-sealed and further incubated for 48–96 h at room temperature. Next, crystals are harvested and flashfrozen in liquid nitrogen. A representative X-ray diffraction image is shown, followed by a model of the inhibitor binding pocket. The electron density map of KSR1:MEK1 in complex with AMP-PNP and trametinib is displayed (PDB ID: 7JUX).$

Journal: Methods in enzymology

Article Title: Conformational control and regulation of the pseudokinase KSR via small molecule binding interactions

doi: 10.1016/bs.mie.2022.03.039

Figure Lengend Snippet: Purification of the KSR1:MEK1 complex for biochemical and structural analysis. (A) Copurification of the KSR1 pseudokinase domain and MEK1 kinase domain is shown as an example (Khan et al., 2020). KSR1 [residues 591–899] and MEK1 [residues 33–393] were coexpressed as His-tagged proteins using the Sf21 insect cell system. (B) Cobaltaffinity purification of KSR1-MEK1 complex using the gravity-flow column (left);the corresponding 8–16% SDS-gel is shown on the right. (C) Partially-purified KSR1:MEK1 complex is further purified using anion exchange chromatography to remove excessive MEK. Several impurities are detected on the 8–16% SDS gel (below). (D) KSR1-MEK1 (1:1) complex is obtained after size-exclusion chromatography. Either the KSR1:MEK1 complex can be isolated as His-tagged protein (left) or KSR1-MEK1 can be treated with trypsin (1:1000, incubated overnight at 4 °C) to remove partially unfolded proteins and flexible loops, and hence highly homogenous proteins are obtained for crystallization. SDS gels are shown below, and the protein fractions corresponding to the tetrameric peak (150 kDa—see Fig. 2 for a tetrameric assembly) are pooled, and concentrated for subsequent analysis. (E) The flow-chart is provided to illustrate the procedure for determining structures of the KSR1:MEK1 complexes bound to interfacial binders, including trametinib (Khan et al., 2020). Similar procedures can be used for solving structures of other MEKi. Cocrystallization of the KSR:MEK1 complex in the presence of orthosteric binders can also be used to solve structures (Brennan et al., 2011; Dhawan, Scopton, & Dar, 2016). (left to right) Crystals of purified KSR1:MEK1 or KSR2:MEK1 complexes are first grown in the presence of 5mM AMP-PNP, and typically first appear within 24 h at room temperature. Crystals of KSR2-MEK1 are shown as an example. Next, individual crystals (72–96 h old) are transferred into a fresh drop containing crystallization solution (20% PEG2000, 200mM Mg acetate, 100mM MES pH 6.5) supplemented with 5 mM AMP-PNP and 1 mM trametinib. The drop is re-sealed and further incubated for 48–96 h at room temperature. Next, crystals are harvested and flashfrozen in liquid nitrogen. A representative X-ray diffraction image is shown, followed by a model of the inhibitor binding pocket. The electron density map of KSR1:MEK1 in complex with AMP-PNP and trametinib is displayed (PDB ID: 7JUX).

Techniques: Purification, Copurification, SDS-Gel, Chromatography, Size-exclusion Chromatography, Isolation, Incubation, Crystallization Assay, Binding Assay

An assay for orthosteric binders at the ATP-binding pocket of purified KSR-MEK complexes. (A) Putative structure of RAF (blue)–KSR (green)–MEK (red) complex based on structural and functional studies (Brennan et al., 2011; Burack & Shaw, 2000; Dhawan et al., 2016; Khan et al., 2020; Lavoie et al., 2018; Michaud et al., 1997; Morrison, 2001; Nguyen et al., 2002; Rajakulendran et al., 2009; Roy, Laberge, Douziech, Ferland-McCollough, & Therrien, 2002; Therrien et al., 1996). KSR mutations that suppress oncogenic RAS signaling (shown in red) localized around the ATP-binding pocket (yellow) as well as the RAF and MEK interfaces (Dhawan et al., 2016). (B) Structure of desthiobiotin-ATP: ATP (left) linker (middle) desthiobiotin (right). (C) Schematic for labeling of the orthosteric sites of KSR and MEK with ATP-biotin in the presence and absence of competing ligands probe. (D) Representative western blot of recombinant MAPK proteins (MEK1, BRAF, KSR1:MEK, and KSR2:MEK) labeled with increasing concentrations of the ATP-biotin probe. (E) Probe-labeled kinases (MEK, BRAF, KSR1:MEK, and KSR2: MEK) treated with increasing concentrations of inhibitors (5, 10 and 20 μM) were measured via western blotting. 2.5 μM of probe was used to label each protein. DMSO was used as a negative control (No inhibitor) and free ATP (100, 1000, and 10,000 μM) was used as a positive control for blocking labeling. (F) The KSR2-MEK1 complex with APS-2-79 identified the inhibitor bound in the orthosteric site of KSR2 (Dhawan et al., 2016).

Journal: Methods in enzymology

Article Title: Conformational control and regulation of the pseudokinase KSR via small molecule binding interactions

doi: 10.1016/bs.mie.2022.03.039

Figure Lengend Snippet: An assay for orthosteric binders at the ATP-binding pocket of purified KSR-MEK complexes. (A) Putative structure of RAF (blue)–KSR (green)–MEK (red) complex based on structural and functional studies (Brennan et al., 2011; Burack & Shaw, 2000; Dhawan et al., 2016; Khan et al., 2020; Lavoie et al., 2018; Michaud et al., 1997; Morrison, 2001; Nguyen et al., 2002; Rajakulendran et al., 2009; Roy, Laberge, Douziech, Ferland-McCollough, & Therrien, 2002; Therrien et al., 1996). KSR mutations that suppress oncogenic RAS signaling (shown in red) localized around the ATP-binding pocket (yellow) as well as the RAF and MEK interfaces (Dhawan et al., 2016). (B) Structure of desthiobiotin-ATP: ATP (left) linker (middle) desthiobiotin (right). (C) Schematic for labeling of the orthosteric sites of KSR and MEK with ATP-biotin in the presence and absence of competing ligands probe. (D) Representative western blot of recombinant MAPK proteins (MEK1, BRAF, KSR1:MEK, and KSR2:MEK) labeled with increasing concentrations of the ATP-biotin probe. (E) Probe-labeled kinases (MEK, BRAF, KSR1:MEK, and KSR2: MEK) treated with increasing concentrations of inhibitors (5, 10 and 20 μM) were measured via western blotting. 2.5 μM of probe was used to label each protein. DMSO was used as a negative control (No inhibitor) and free ATP (100, 1000, and 10,000 μM) was used as a positive control for blocking labeling. (F) The KSR2-MEK1 complex with APS-2-79 identified the inhibitor bound in the orthosteric site of KSR2 (Dhawan et al., 2016).

Techniques: Binding Assay, Purification, Functional Assay, Labeling, Western Blot, Recombinant, Negative Control, Positive Control, Blocking Assay

Developing NanoBRET to measure the pharmacology of MEK inhibitors on KSR–MEK complexes in live cells. (A) Structures of Trametinib and the NanoBRET probe we call Tram-bo. Within Tram-bo, the parent compound is shown in black, the linker portion is green, and Bodipy is red. (B) Nanoluciferase (−NL) placement on MEK or KSR allows pharmacology to be measured on MEK and the MEK-KSR complex in model systems (e.g., HEK293 cells). Shown on the left is a cartoon for measuring Tram-bo bound to MEK1-NL. Also shown are putative complexes of Tram-bo bound to MEK-KSR formed through MEK1-NL coexpressed with either KSR1 or KSR2 (middle) or from the expression of KSR1-NL or KSR2-NL, which is expected to form complexes with endogenous MEK (far right). (C) Build-up curves to measure the apparent EC50 of Tram-bo on MEK and the MEK–KSR complex. (D) Order of addition matters for tracers that have nanomolar apparent EC50 values as seen for Trametinib on MEK-Nanoluciferase. (E) Steady-state dose-response and washout experiments depend on specific interactions at the interface between MEK and KSR1 as determined by the use of a KSR1 mutant, W781D. (F) Specificity of KSR1-NL assays are evidenced by the lack of a Trametinib dose-response using the W781D mutant. Please see Fig. 2 for the location of the W781 residue at helix αG and at the interface of the KSR1-MEK1 complex. Data in panels (E and F) reproduced from Khan, Zaigham M., Alexander M. Real, William M. Marsiglia, Arthur Chow, Mary E. Duffy, Jayasudhan R. Yerabolu, Alex P. Scopton, and Arvin C. Dar. 2020. “Structural basis for the action of the drug trametinib at KSR-bound MEK.” Nature 588 (7838): 509–14.

Journal: Methods in enzymology

Article Title: Conformational control and regulation of the pseudokinase KSR via small molecule binding interactions

doi: 10.1016/bs.mie.2022.03.039

Figure Lengend Snippet: Developing NanoBRET to measure the pharmacology of MEK inhibitors on KSR–MEK complexes in live cells. (A) Structures of Trametinib and the NanoBRET probe we call Tram-bo. Within Tram-bo, the parent compound is shown in black, the linker portion is green, and Bodipy is red. (B) Nanoluciferase (−NL) placement on MEK or KSR allows pharmacology to be measured on MEK and the MEK-KSR complex in model systems (e.g., HEK293 cells). Shown on the left is a cartoon for measuring Tram-bo bound to MEK1-NL. Also shown are putative complexes of Tram-bo bound to MEK-KSR formed through MEK1-NL coexpressed with either KSR1 or KSR2 (middle) or from the expression of KSR1-NL or KSR2-NL, which is expected to form complexes with endogenous MEK (far right). (C) Build-up curves to measure the apparent EC50 of Tram-bo on MEK and the MEK–KSR complex. (D) Order of addition matters for tracers that have nanomolar apparent EC50 values as seen for Trametinib on MEK-Nanoluciferase. (E) Steady-state dose-response and washout experiments depend on specific interactions at the interface between MEK and KSR1 as determined by the use of a KSR1 mutant, W781D. (F) Specificity of KSR1-NL assays are evidenced by the lack of a Trametinib dose-response using the W781D mutant. Please see Fig. 2 for the location of the W781 residue at helix αG and at the interface of the KSR1-MEK1 complex. Data in panels (E and F) reproduced from Khan, Zaigham M., Alexander M. Real, William M. Marsiglia, Arthur Chow, Mary E. Duffy, Jayasudhan R. Yerabolu, Alex P. Scopton, and Arvin C. Dar. 2020. “Structural basis for the action of the drug trametinib at KSR-bound MEK.” Nature 588 (7838): 509–14.

Techniques: Expressing, Mutagenesis

Review

Structured Review

Images

Similar Products

Image Search Results