R. palustris TIE-1 was grown anaerobically on YP medium (10 g/L yeast extract and 20 g/L peptone) at 30 °C under a 36-W white fluorescent lamp for 5 days before the cells were harvested using centrifugation and stored at −80 °C. The cells were thawed and resuspended in 20 mM Tris-HCl pH 8 before a three-passage disruption using a French press at 1000 psi. The lysate was then ultracentrifuged at 204,709× g for 90 min, at 4 °C, and the membrane pellets were solubilized using 20 mM Tris-HCl pH 8 with 1% SB-12 (Sulfobetaine-12) and stirred for 1 h at room temperature before the second ultracentrifugation at 204,709× g at 4 °C. The supernatant was loaded into a DEAE Sepharose, and the fraction containing LH-RC was eluted with 300 mM NaCl. The purity of the LH-RC was confirmed using SDS-PAGE with Blue Safe staining (NZYTech, Lisboa, Portugal) and UV-visible spectroscopy. An extinction coefficient of 803 nm (ε800nm = 2,570,000 M−1cm−1) for the bacteriochlorophyll a pigments was used for quantification purposes [40 (link)].
Blue safe staining
Manufactured by NZYTech
Sourced in Portugal
Blue Safe staining is a laboratory equipment product designed for staining biological samples. It provides a reliable and consistent method for visualizing specific components or structures within the samples.
Automatically generated - may contain errors
Lab products found in correlation
Histrap ff crude column, by GE Healthcare (2 mentions)
Akta purifier, by GE Healthcare (1 mentions)
Ananodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)
P nitrophenyl d galactopyranoside pnp gal, by Merck Group (1 mentions)
Isopropyl β d 1 thiogalactopyranoside iptg, by Merck Group (1 mentions)
Lambda protein phosphatase, by New England Biolabs (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
5 protocols using blue safe staining
1
Purification and Characterization of Light-Harvesting Reaction Center from R. palustris
2
Purification of Recombinant Xylanase from E. coli
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Cell crude extracts were prepared from E. coli cultures grown at 37 °C up to a cell density (OD600) of 0.6 and induced with 1 mM IPTG, either at 16 °C overnight or 37 °C for 5 h. The cells were disrupted by sonication in buffer A (20 mM phosphate buffer, pH 7.4, 10 mM imidazole, 500 mM NaCl). Protein extracts were recovered by centrifugation at 12,000×g during 25 min and subjected to nickel-affinity chromatography using 1 mL HisTrap FF crude column (GE Healthcare) mounted in AKTA-Purifier (GE), using buffer B (20 mM phosphate buffer, pH 7.4, 500 mM imidazole, 500 mM NaCl) for elution. Eluted fractions showing xylanase activity were dialyzed against buffer C (20 mM Tris–HCl, pH 7 50 mM NaCl). The protein was analyzed by SDS-PAGE, using Blue Safe staining (Nzytech). An image of the gel was taken with a Proxima AQ-4 gel documentation system (Isogen) and the resulting amount of protein in the gel bands was quantified using FIJI software [52 (link)].
3
Recombinant Xylanase Purification Protocol
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Cell crude extracts were obtained from E.
coli cultures grown at 37 °C until reaching
OD600 of 0.6 and then were induced with 1 mM IPTG,
either at 37 °C for 5 h or 16 °C overnight. Buffer A (20 mM phosphate
buffer, pH 7.4, 10 mM imidazole, 500 mM NaCl) was used to disrupt the cells
by sonication. Protein extracts were obtained by centrifugation at 12,000 g
during 25 min. Protein purification was performed using nickel affinity
chromatography with 1 mL HisTrap FF crude column (GE Healthcare) mounted in
AKTA-Purifier (GE). Buffer B (20 mM phosphate buffer, pH 7.4, 500 mM
imidazole, 500 mM NaCl) was used for elution. Eluted fractions with xylanase
activity were dialyzed against buffer C (20 mM Tris-HCl, pH 7.0, 50 mM
NaCl). Protein concentration in the eluted fractions was determined in a
NanoDrop spectrophotometer (Thermo Fisher). The purity of the protein
recovered was analyzed by SDS-PAGE, using Blue Safe staining (Nzytech). An
image of the gel was taken with a Proxima AQ-4 gel documentation system
(Isogen) and the amount of protein in the gel bands was quantified using
FIJI software [20] (link).
coli cultures grown at 37 °C until reaching
OD600 of 0.6 and then were induced with 1 mM IPTG,
either at 37 °C for 5 h or 16 °C overnight. Buffer A (20 mM phosphate
buffer, pH 7.4, 10 mM imidazole, 500 mM NaCl) was used to disrupt the cells
by sonication. Protein extracts were obtained by centrifugation at 12,000 g
during 25 min. Protein purification was performed using nickel affinity
chromatography with 1 mL HisTrap FF crude column (GE Healthcare) mounted in
AKTA-Purifier (GE). Buffer B (20 mM phosphate buffer, pH 7.4, 500 mM
imidazole, 500 mM NaCl) was used for elution. Eluted fractions with xylanase
activity were dialyzed against buffer C (20 mM Tris-HCl, pH 7.0, 50 mM
NaCl). Protein concentration in the eluted fractions was determined in a
NanoDrop spectrophotometer (Thermo Fisher). The purity of the protein
recovered was analyzed by SDS-PAGE, using Blue Safe staining (Nzytech). An
image of the gel was taken with a Proxima AQ-4 gel documentation system
(Isogen) and the amount of protein in the gel bands was quantified using
FIJI software [20] (link).
4
Nanoflowers Formation and Characterization
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Salts used for nanoflowers formation (ZnSO4, CaCl2, MgCl2, MnSO4, CoCl2, CuSO4) and for buffer preparation (NaCl and MgCl2) and lactose 1-hydrate were from Panreac Química (Spain). Chromogenic -galactosidase substrate, p-nitrophenyl ß-D-Galactopyranoside (pNP-Gal) and isopropyl β-D-1-thiogalactopyranoside (IPTG) were purchased from Sigma Aldrich.
Protein in SDS-PAGE analysis was stained with BlueSafe Staining from NZYTech.
Protein in SDS-PAGE analysis was stained with BlueSafe Staining from NZYTech.
5
Cell Lysate Preparation and Protein Analysis
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To prepare lysates cells pellets were incubated in lysis buffer 30 min on ice (20 mM Tris-HCl pH 7.5-8, 137 mM NaCl, 1 mM EDTA, 1.5 mM MgCl2, 10% glycerol, 1% TritonX100). Lysates were then clarified by centrifugation 15 min at 13,200 rpm and 4°C on a table top centrifuge. Protein concentration was measured using the Bradford protein assay (Bio-Rad). When indicated, cell lysates were supplemented with Lambda Protein Phosphatase (P0753S, NEB) at a concentration of 200 units/50 μl reaction during 30 min at 30°C. For western blots, 30 to 40 μg of protein extract were loaded per lane for SDS-PAGE (8% acrylamide concentration). Coomassie staining was performed using BlueSafe staining (MB15201, NZYTECH). All gels were analyzed with the Odyssey Infrared imaging system (Li-Cor).
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