Bio-Rad gravity fl ow chromatography

Fig. 1 The principle of calmodulin-afﬁ nity <t>chromatography.</t> ( a ) Detergent - solubilized plasma membrane proteins are applied to a column packed with calmodulin coupled to Sepharose 4B, in the presence of 0.1 mM CaCl 2 . Calmodulin interacts reversibly with the PMCA molecules in the presence of Ca 2+ . ( b ) Proteins binding calmodulin with high afﬁ nity, such as the PMCA, will be retained on the column, as they interact with the immobilized calmodulin. Non-binding proteins are eluted by a washing of the column with a Ca 2+ -containing buffer. ( c ) Proteins binding speciﬁ cally and reversibly to calmodulin, such as the PMCA, can now be eluted from the column by exchanging the Ca 2+ -containing buffer for a Ca 2+ -free buffer containing EDTA

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Fig. 1 The principle of calmodulin-afﬁ nity chromatography. ( a ) Detergent - solubilized plasma membrane proteins are applied to a column packed with calmodulin coupled to Sepharose 4B, in the presence of 0.1 mM CaCl 2 . Calmodulin interacts reversibly with the PMCA molecules in the presence of Ca 2+ . ( b ) Proteins binding calmodulin with high afﬁ nity, such as the PMCA, will be retained on the column, as they interact with the immobilized calmodulin. Non-binding proteins are eluted by a washing of the column with a Ca 2+ -containing buffer. ( c ) Proteins binding speciﬁ cally and reversibly to calmodulin, such as the PMCA, can now be eluted from the column by exchanging the Ca 2+ -containing buffer for a Ca 2+ -free buffer containing EDTA

Journal: Methods in Molecular Biology

Article Title: P-Type ATPases

doi: 10.1007/978-1-4939-3179-8

Figure Lengend Snippet: Fig. 1 The principle of calmodulin-afﬁ nity chromatography. ( a ) Detergent - solubilized plasma membrane proteins are applied to a column packed with calmodulin coupled to Sepharose 4B, in the presence of 0.1 mM CaCl 2 . Calmodulin interacts reversibly with the PMCA molecules in the presence of Ca 2+ . ( b ) Proteins binding calmodulin with high afﬁ nity, such as the PMCA, will be retained on the column, as they interact with the immobilized calmodulin. Non-binding proteins are eluted by a washing of the column with a Ca 2+ -containing buffer. ( c ) Proteins binding speciﬁ cally and reversibly to calmodulin, such as the PMCA, can now be eluted from the column by exchanging the Ca 2+ -containing buffer for a Ca 2+ -free buffer containing EDTA

Article Snippet: Two 10 cm × 0.5 cm (inner diameter) Glass Econo-Column ® columns for gravity fl ow chromatography (Bio-Rad, Hercules, CA, USA) with outlet tubing of 1 and 2 mL dead-volume, respectively.

Techniques: Chromatography, Clinical Proteomics, Membrane, Binding Assay

Fig. 1 Relative concentration of fatty acid species before and after LEC of a SERCA preparation. The relative concentrations of fatty acid species determined by gas chromatography of methylated fatty acids from extracted samples show a strong enrichment to ~85 % target lipid after LEC. The following samples were analyzed: DOC SR, deoxycholate-extracted sarcoplasmic reticulum (SR) micro- somes before solubilization; Solub. SR, C 12 E 8 solubilized SR microsomes enriched in SERCA; DOPC exchange, solubilized SERCA after LEC with DOPC as target lipid; DOPC strip, DOPC exchanged SERCA after sucrose step-gradient spin to strip the sample from bulk lipids. Error bars correspond to the standard deviation of results from three independent experiments. Adapted ﬁ gure from [ 8 ]

Journal: Methods in Molecular Biology

Article Title: P-Type ATPases

doi: 10.1007/978-1-4939-3179-8

Figure Lengend Snippet: Fig. 1 Relative concentration of fatty acid species before and after LEC of a SERCA preparation. The relative concentrations of fatty acid species determined by gas chromatography of methylated fatty acids from extracted samples show a strong enrichment to ~85 % target lipid after LEC. The following samples were analyzed: DOC SR, deoxycholate-extracted sarcoplasmic reticulum (SR) micro- somes before solubilization; Solub. SR, C 12 E 8 solubilized SR microsomes enriched in SERCA; DOPC exchange, solubilized SERCA after LEC with DOPC as target lipid; DOPC strip, DOPC exchanged SERCA after sucrose step-gradient spin to strip the sample from bulk lipids. Error bars correspond to the standard deviation of results from three independent experiments. Adapted ﬁ gure from [ 8 ]

Techniques: Concentration Assay, Gas Chromatography, Methylation, Stripping Membranes, Standard Deviation

$Fig. 1 Chromatograms from size exclusion chromatography of the nanodisc preparation. (a) Chromatograms of first run. (b) First run, zoom. (c) Second run of the fraction corresponding to one NKA molecule per nanodisc. (d) Second run of the fraction corresponding to two NKA molecules per nanodisc$

Journal: Methods in Molecular Biology

Article Title: P-Type ATPases

doi: 10.1007/978-1-4939-3179-8

Figure Lengend Snippet: Fig. 1 Chromatograms from size exclusion chromatography of the nanodisc preparation. (a) Chromatograms of first run. (b) First run, zoom. (c) Second run of the fraction corresponding to one NKA molecule per nanodisc. (d) Second run of the fraction corresponding to two NKA molecules per nanodisc

Techniques: Size-exclusion Chromatography

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