bio spin p 30 gel filtration spin columns  (Bio-Rad)

Journal: STAR Protocols

Article Title: Protocol to generate, purify, and analyze antibody-oligonucleotide conjugates from off-the-shelf antibodies

doi: 10.1016/j.xpro.2024.103329

Figure Lengend Snippet:

Article Snippet: Bio-Spin P-30 gel filtration spin columns , Bio-Rad , 7326232.

Techniques: Recombinant, Synthesized, Bicinchoninic Acid Protein Assay, Software, Spectrophotometry, Filtration, Chromatography, Mass Spectrometry

Journal: eLife

Article Title: A single power stroke by ATP binding drives substrate translocation in a heterodimeric ABC transporter

doi: 10.7554/eLife.55943

Figure Lengend Snippet: ( a ) Experimental scheme monitoring the OF-to-IF transition. ( b ) Nucleotide occlusion after the IF-to-OF transition. TmrA EQ B in Nds (2 µM) was incubated for 5 min at 45 °C with Mg 2+ -ATP (1 mM, traced with [α 32 P]-ATP or [γ 32 P]-ATP). ATP (2 mM) was added and unbound nucleotides were removed by rapid gel filtration. The radiolabeled nucleotides occluded by TmrA EQ B were identified by thin layer chromatography and autoradiography. Representative autoradiograms for three independent triplicates are shown. ( c ) ATPase activity. TmrA EQ B (1 µM) was incubated with Mg 2+ -ATP (1 mM, traced with [γ 32 P]-ATP) for 5 min at 45 °C and 15 min at 20 °C. Control reactions were performed in the presence of EDTA (10 mM). ATP autohydrolysis was recorded in the absence of TmrA EQ B. The release of [γ 32 P] was quantified by thin layer chromatography and autoradiography. Data were normalized to autohydrolysis and the mean values ± SD (n = 3) are displayed. ( d ) Nucleotide dissociation upon the OF-to-IF return. TmrA EQ B in Nds (0.2 µM) was incubated with Mg 2+ -ATP (3 µM, traced with 3 H-ATP) for 5 min at 45 °C. Unbound nucleotides were removed by rapid gel filtration. An excess of Mg 2+ -ATP (1 mM) was added to prevent any reassociation of released nucleotides. Nucleotide dissociation was followed by SPA at 20 °C. The mean ± SD (n = 3) is displayed and monoexponentially fitted, leading to a dissociation rate constant k off of 0.029 ± 0.002 min −1 (τ 1/2 = 21–26 min 95% confidence interval). ( e ) Peptide rebinding along the OF-to-IF return. TmrA EQ B (0.2 µM) complexes were converted to the OF state as described in a ). The excess of ATP was removed by rapid gel filtration. R9LQK peptide binding (10 µM, traced with 3 H-R9L) was monitored by SPA at 20 °C. The mean ± SD (n = 3) is displayed and monoexponentially fitted, leading to an apparent association rate k on of 0.030 ± 0.001 min −1 (τ 1/2 = 21–25 min 95% confidence interval). ( f ) Release of inorganic phosphate along the OF-to-IF return. TmrA EQ B in Nds (2 µM) was incubated with Mg 2+ -ATP (1 mM, traced with [γ 32 P]-ATP) and converted to the OF state as described in b ). ATP (2 mM) was added and unbound nucleotides were removed by rapid gel filtration. Subsequently, a large excess of unlabeled ATP (2 mM) was added to prevent reassociation of [γ 32 P]-ATP and thus to guarantee a single-turnover round of ATP hydrolysis. Nucleotides and released phosphate were analyzed as in panel c ). It is important to note that some ATP is hydrolyzed before the TLC analysis. The mean ± SD (n = 3) is displayed and monoexponentially fitted, leading to inversely correlated rates of ATP turnover and phosphate release ( k on ( 32 P i )= k off ([γ 32 P]-ATP)=0.022 ± 0.005 min −1 ). Figure 2—source data 1. Source Data to and .

Article Snippet: If indicated, the excess of free nucleotides was removed by rapid gel filtration (Bio-Spin columns P-30, Bio-Rad).

Techniques: Incubation, Filtration, Thin Layer Chromatography, Autoradiography, Activity Assay, Control, Binding Assay

Journal: eLife

Article Title: A single power stroke by ATP binding drives substrate translocation in a heterodimeric ABC transporter

doi: 10.7554/eLife.55943

Figure Lengend Snippet: ( a ) Nucleotide occlusion at different temperatures and subsequent dissociation. TmrA EQ B in Nds (0.2 µM) was incubated with Mg 2+ -ATP (3 µM, traced with 3 H-ATP) for 5 min at 20 °C or 4°C. Unbound nucleotides were removed by rapid gel filtration. Subsequently, ATP (1 mM) was added to prevent any reassociation of released ATP. Nucleotide dissociation was followed by SPA at 20 °C. Data shown as mean ± SD (n = 3) are monoexponentially fitted, leading to a dissociation rate constant k off of 0.032 ± 0.002 min −1 (τ 1/2 = 19–24 min 95% confidence interval). ( b ), Nucleotide dissociation at 4 °C. TmrA EQ B (0.2 µM) was incubated with Mg 2+ -ATP for 5 min at 45°C as described in a ). After rapid gel filtration and ATP addition, nucleotide dissociation was followed by SPA at 4 °C. The mean ± SD (n = 3) is displayed and monoexponentially fitted, leading to a dissociation rate constant k off of 0.021 ± 0.001 min −1 (τ 1/2 = 30–38 min 95% confidence interval). ( c ), Nucleotide dissociation at 45 °C. TmrA EQ B in Nds (0.2 µM) was incubated with Mg 2+ -ATP for 5 min at 45 °C as described in a ). After rapid gel filtration and ATP addition, nucleotide dissociation was followed by SPA at 45 °C. The mean ± SD (n = 3) is displayed and monoexponentially fitted, leading to a dissociation rate constant k off of 0.036 ± 0.003 min −1 (τ 1/2 = 16–24 min 95% confidence interval).

Article Snippet: If indicated, the excess of free nucleotides was removed by rapid gel filtration (Bio-Spin columns P-30, Bio-Rad).

Techniques: Incubation, Filtration