A synthetic cDNA for human CYP17A1 was modified to delete residues 2–19, substitute the hydrophilic sequence 20RRCP23 (link) with 20AKKT23 (link), and add a C-terminal four histidine tag (fig. S6 ) before cloning into the pCWori+ plasmid and overexpression in E. coli JM109 cells. Protein was purified by nickel affinity, cation exchange, and size exclusion chromatography. Abiraterone was synthesized (Methods). Binding affinities were determined using a UV/vis spectral shift assay. Progesterone 17α-hydroxylation was evaluated using HPLC separation and UV detection. For crystallography, inhibitors were included throughout purification. Crystals were grown from CYP17A1 (30 mg/mL) complexed with inhibitor using hanging-drop vapor diffusion to equilibrate against 30% PEG 3350, 0.175 M Tris, pH 8.5, 0.30 M ammonium sulfate, and 3% glycerol. Diffraction data was collected and phased by molecular replacement. Iterative model building and refinement generated the final model. Substrates were docked using Surflex-Dock30 (link).
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Nickel sulfate
Nickel sulfate
Nickel sulfate is an inorganic compound composed of nickel and sulfate ions.
It is a pale green crystalline solid that is soluble in water and widely used in various industrial processes and applications.
Nickel sulfate plays a crucial role in electroplating, pigment manufacturing, and catalytic reactions.
It is also employed in the production of nickel-based alloys and batteries.
Researchers studying nickel sulfate may leverage PubCompare.ai's AI-driven platform to enhance the reproducibility and accuracy of their studies.
The platform can effortlessly locate optimized protocols from literature, preprints, and patents, while AI-powered comparisons help identify the best methods and products.
This data-driven approach supports informed decision making in nickel sulfate resesarch.
It is a pale green crystalline solid that is soluble in water and widely used in various industrial processes and applications.
Nickel sulfate plays a crucial role in electroplating, pigment manufacturing, and catalytic reactions.
It is also employed in the production of nickel-based alloys and batteries.
Researchers studying nickel sulfate may leverage PubCompare.ai's AI-driven platform to enhance the reproducibility and accuracy of their studies.
The platform can effortlessly locate optimized protocols from literature, preprints, and patents, while AI-powered comparisons help identify the best methods and products.
This data-driven approach supports informed decision making in nickel sulfate resesarch.
Most cited protocols related to «Nickel sulfate»
abiraterone
Biological Assay
Cells
Crystallography
CYP17A1 protein, human
Diffusion
DNA, Complementary
Escherichia coli
Gel Chromatography
Glycerin
High-Performance Liquid Chromatographies
Histidine
Homo sapiens
Hydroxylation
inhibitors
Nickel
Plasmids
polyethylene glycol 3350
Progesterone
Proteins
Sulfate, Ammonium
Tromethamine
Brain
Heparin
Immunoperoxidase Techniques
In Situ Hybridization
Mice, Laboratory
Nickel
Peroxide, Hydrogen
RNA, Messenger
Serum
Serum Albumin, Bovine
sodium borohydride
Sulfates, Inorganic
Tissues
Protein Expression and Purification—The pRSETa vector
containing GCaMP2 was a kind gift of Karel Svoboda (Janelia Farm Research
Campus, Howard Hughes Medical Institute). GCaMP2 was expressed and purified as
described previously (17 ).
Briefly, BL21(DE3) cells containing pRSETa harboring gcamp2 or
gcamp2 mutants were grown in ZYM-5052 medium
(18 ) for 48 h at 25 °C
with shaking at 200 rpm. After centrifugation, cell lysis, and clarification,
proteins were purified from the cell-free extract by nickel-affinity
chromatography. Protein purity over 95% was confirmed by SDS-PAGE analysis.
Proteins were dialyzed into 20 mm Tris-HCl, 100 mm NaCl,
2 mm CaCl2, pH 8.0, and concentrated. Calcium-free
samples were prepared identically, except the buffer contained 5 mm EGTA instead of CaCl2.
Crystallization and Data Collection—All GCaMP
crystallization was carried out at 20 °C. All GCaMP protein samples for
crystallization were in 20 mm Tris, 100 mm NaCl, 2
mm CaCl2, pH 8.0, except for the 8EF-apo mutant where
the same buffer with 5 mm EGTA substituted for CaCl2 was
used. All crystals used for data collection were grown using the hanging-drop
vapor diffusion method in 24-well VDX plates. Crystallization of
Ca2+-saturated dimeric GCaMP2 was described previously
(17 ). The calcium-saturated
K378W (at 5.6 mg/ml) and G87R (at 1.5 mg/ml) mutants crystallized ∼4 days
after mixing with a precipitant solution consisting of 0.1m magnesium formate dihydrate and 15% polyethylene glycol 3,350 using drop
ratios of 2 μl of protein to 2 μl of precipitant for K378W and 1.5-2.5
μl for G87R. Ca2+-saturated monomeric GCaMP2 was crystallized
identically to the K378W and G87R mutants using a drop ratio of 2 μl to 2
μl except that the drops were microseeded by streak seeding from K378W
crystals immediately following setup. These crystals required more than 4
weeks to grow and had a distinct morphology. 8EF-apo GCaMP2 was crystallized
after 1 week by mixing 2 μl of protein solution (9.5 mg/ml) with 2 μl of
a precipitant solution consisting of 0.2m lithium sulfate
monohydrate, 0.1m BisTris, pH 5.5, and 25% polyethylene glycol
3,350.
All crystals were cryoprotected for data collection by quickly (<10 s)
soaking in the precipitant solution supplemented with 20% glycerol and then
mounted in a nitrogen gas stream at 100 K or plunged into liquid nitrogen for
storage and transport to synchrotron beamlines. All data were collected at 100
K in a N2 gas stream. X-ray diffraction data for the G87R mutant
was collected in-house on a Rigaku RU-H3R rotating copper anode x-ray
generator, equipped with a Saturn 92 CCD detector and X-stream 2000
low-temperature system. Data for Ca2+-saturated monomeric GCaMP2
was collected at the Advanced Light Source, beamline 8.2.2. Diffraction data
from crystals of Ca2+-dimer, K378W, and 8EF-apo GCaMP2 were
collected at the Advance Photon Source, beamline 31-ID.
X-ray diffraction data for G87R were integrated and scaled using
d*TREK (19 (link)) from
within the CrystalClear software package (Rigaku/Molecular Structure
Corporation, Woodlands, TX). Data for Ca2+-saturated monomeric
GCaMP2 were integrated and scaled in HKL2000
(20 ). Data from crystals of
Ca2+-dimer, K378W, and 8EF-apo GCaMP2 were processed using Mosflm
(21 ) and Scala
(22 (link)).
Structure Solution, Model Building, and Refinement—All
GCaMP2 structures were solved by molecular replacement using the program
Phaser (23 ). The
Ca2+-saturated dimer structure was solved as described previously
(17 ) using the published
coordinates of GFP (Protein Data Bank (PDB) entry 1EMA) and the coordinates of
M13-bound calmodulin (PDB entry 1CDL) as search models. The G87R
Ca2+-bound monomer mutant structure was solved by searching
sequentially using the cpEGFP domain and CaM-M13 domains from the refined
Ca2+-dimer structure and data between 29.3- and 2.8-Å
resolution. Clear solutions were obtained in space group
P41212 with translation function Z-scores of
43.1 and 21.3, respectively, for the two domains. Strong positive peaks in the
difference map at the expected positions of the calcium ions in CaM (which
were omitted from the MR model) indicated the correctness of the solutions.
The K378W mutant crystals were isomorphous with those of the G87R mutant and
the G87R model was used directly for rigid-body refinement against data from
K378W crystals. The Ca2+-saturated monomeric GCaMP2 structure was
solved using the refined K378W coordinates as a search model. A clear solution
was obtained in space group P21212 with a translation
function Z-score of 39.2 using data between 45.4- and 2.65-Å
resolution. The 8EF-apo GCaMP2 calcium-free mutant structure was solved by
searching for the cpEGFP domain from the Ca2+-dimer structure. A
clear solution was obtained in space group C2 with a translation function
Z-score of 18.9 using data between 31.9- and 2.8-Å resolution.
Subsequently searching for the calcium-free N-terminal or C-terminal lobes of
CaM (PDB code 1CFD (24 (link))) did
not reveal any clear solutions. Some positive difference density was present
in the electron density maps calculated using the cpEGFP domain solution that
suggested the position of the N-terminal lobe of CaM, which was placed
manually into density and refined. The correctness of this CaM N-terminal lobe
placement was indicated by additional positive difference density for the
linker connecting cpEGFP and the CaM N-terminal lobe, which was subsequently
built.
All models were improved by iterative cycles of model building in Coot
(25 ) and positional refinement
in REFMAC (26 ). Final GCaMP2
models have reasonable R-factors and model geometries, as illustrated
inTable 1 . A portion of the
electron density map for each structure is provided in supplemental Fig.
S1.
using a Superdex 200 10/300 GL column (GE Healthcare) at a flow rate of 0.5 ml
min-1 in 20 mm Tris, 100 mm NaCl, 2
mm CaCl2, pH 8.0, for calcium-saturated samples or with
5 mm EGTA in place of CaCl2 for calcium-free samples.
Molecular weights were estimated by comparison with elution volumes of
standard proteins (Bio-Rad).
Sedimentation Velocity Analytical
Ultracentrifugation—Analytical ultracentrifugation of GCaMP2
samples was carried out in a Beckman XL-I analytical ultracentrifuge (Beckman
Coulter, Fullerton, CA) within the Biophysics Instrumentation Facility at the
Massachusetts Institute of Technology. Absorbance scans at 280 nm were
collected on calcium-free (24 μm ) and calcium-saturated (28
μm ) GCaMP2 samples in two-sector cells within a four-hole
AnTi-60 rotor at 42,000 rpm. Data were collected at 20 °C in the same
buffers as the SEC experiments. Absorbance scans were modeled using a
continuous c(s) distribution within Sedfit
(27 (link)), correcting for buffer
density and viscosity and using a partial specific volume of 0.7300
cm3 g-1. Molecular weight of observed species
(Fig. 2B ) was
estimated based on the best-fit frictional ratio as determined by Sedfit for
each sample.
![]()
GCaMP2—To measure intracellular [GCaMP2], acute brain slices
containing neurons expressing GCaMP2 were prepared and imaged as previously
described (6 (link),
16 (link)). Purified GCaMP2 was
diluted into pipette internal solution supplemented with 1 mm K2EGTA at 0.1, 1, and 10 μm concentrations. Each
solution was drawn into a thin glass capillary (ID = 0.02 mm, Vitrocom number
RT5002). Their fluorescence intensities were measured under two-photon
excitation with identical parameters (910 nm excitation) to neuron imaging.
0.1 μm GCaMP2 was not bright enough to significantly exceed PMT
dark current at laser powers used for neuronal imaging.
Intracellular GCaMP2 concentration in neurons with robust fluorescent
responses to action potential firing was estimated by a linear extrapolation
from the purified 10 μm GCaMP2 fluorescence intensity.
Intracellular GCaMP2 was assumed to be in the apo state
(6 (link)).
Generation and Screening of GCaMP2 Mutants—Mutants of GCaMP2
were prepared by site-directed mutagenesis (see supplemental Tables S1-S3) and
confirmed by sequencing. Preliminary screening for variants with altered
oligomerization equilibria (supplemental Table S1) was performed by passing
100-μl aliquots of cell-free extract from 200-ml cultures of overexpressed
GCaMP2 over a Superdex 200 10/300 GL column while monitoring the absorption at
280 and 495 nm.
Spectrophotometric Analysis—Absorbance spectra were obtained
in a Safire2 (Tecan) with UVStar 96-well plates (Greiner) for both
the calcium-free (10 mm EGTA) and calcium-loaded state (10
mm CaCl2). For fluorescence spectra, Fluotrac 200 plates
(Greiner) were used. Samples were diluted 10-fold in zero free calcium buffer
(Invitrogen) (30 mm MOPS, 100 mm KCl, 10 mm EGTA, pH 7.2) for calcium-free spectra, and in 39 μm free
calcium buffer (Invitrogen) (30 mm MOPS, 10 mm Ca-EGTA
in 100 mm KCl, pH 7.2) for calcium-loaded spectra. For absorbance
measurements, samples were dialyzed into 20 mm Tris, 100
mm NaCl, and EGTA or CaCl2 was added to a final
concentration of 10 mm .
containing GCaMP2 was a kind gift of Karel Svoboda (Janelia Farm Research
Campus, Howard Hughes Medical Institute). GCaMP2 was expressed and purified as
described previously (17 ).
Briefly, BL21(DE3) cells containing pRSETa harboring gcamp2 or
gcamp2 mutants were grown in ZYM-5052 medium
(18 ) for 48 h at 25 °C
with shaking at 200 rpm. After centrifugation, cell lysis, and clarification,
proteins were purified from the cell-free extract by nickel-affinity
chromatography. Protein purity over 95% was confirmed by SDS-PAGE analysis.
Proteins were dialyzed into 20 m
2 m
samples were prepared identically, except the buffer contained 5 m
Crystallization and Data Collection—All GCaMP
crystallization was carried out at 20 °C. All GCaMP protein samples for
crystallization were in 20 m
m
the same buffer with 5 m
used. All crystals used for data collection were grown using the hanging-drop
vapor diffusion method in 24-well VDX plates. Crystallization of
Ca2+-saturated dimeric GCaMP2 was described previously
(17 ). The calcium-saturated
K378W (at 5.6 mg/ml) and G87R (at 1.5 mg/ml) mutants crystallized ∼4 days
after mixing with a precipitant solution consisting of 0.1
ratios of 2 μl of protein to 2 μl of precipitant for K378W and 1.5-2.5
μl for G87R. Ca2+-saturated monomeric GCaMP2 was crystallized
identically to the K378W and G87R mutants using a drop ratio of 2 μl to 2
μl except that the drops were microseeded by streak seeding from K378W
crystals immediately following setup. These crystals required more than 4
weeks to grow and had a distinct morphology. 8EF-apo GCaMP2 was crystallized
after 1 week by mixing 2 μl of protein solution (9.5 mg/ml) with 2 μl of
a precipitant solution consisting of 0.2
monohydrate, 0.1
3,350.
All crystals were cryoprotected for data collection by quickly (<10 s)
soaking in the precipitant solution supplemented with 20% glycerol and then
mounted in a nitrogen gas stream at 100 K or plunged into liquid nitrogen for
storage and transport to synchrotron beamlines. All data were collected at 100
K in a N2 gas stream. X-ray diffraction data for the G87R mutant
was collected in-house on a Rigaku RU-H3R rotating copper anode x-ray
generator, equipped with a Saturn 92 CCD detector and X-stream 2000
low-temperature system. Data for Ca2+-saturated monomeric GCaMP2
was collected at the Advanced Light Source, beamline 8.2.2. Diffraction data
from crystals of Ca2+-dimer, K378W, and 8EF-apo GCaMP2 were
collected at the Advance Photon Source, beamline 31-ID.
X-ray diffraction data for G87R were integrated and scaled using
d*TREK (19 (link)) from
within the CrystalClear software package (Rigaku/Molecular Structure
Corporation, Woodlands, TX). Data for Ca2+-saturated monomeric
GCaMP2 were integrated and scaled in HKL2000
(20 ). Data from crystals of
Ca2+-dimer, K378W, and 8EF-apo GCaMP2 were processed using Mosflm
(21 ) and Scala
(22 (link)).
Structure Solution, Model Building, and Refinement—All
GCaMP2 structures were solved by molecular replacement using the program
Phaser (23 ). The
Ca2+-saturated dimer structure was solved as described previously
(17 ) using the published
coordinates of GFP (Protein Data Bank (PDB) entry 1EMA) and the coordinates of
M13-bound calmodulin (PDB entry 1CDL) as search models. The G87R
Ca2+-bound monomer mutant structure was solved by searching
sequentially using the cpEGFP domain and CaM-M13 domains from the refined
Ca2+-dimer structure and data between 29.3- and 2.8-Å
resolution. Clear solutions were obtained in space group
P41212 with translation function Z-scores of
43.1 and 21.3, respectively, for the two domains. Strong positive peaks in the
difference map at the expected positions of the calcium ions in CaM (which
were omitted from the MR model) indicated the correctness of the solutions.
The K378W mutant crystals were isomorphous with those of the G87R mutant and
the G87R model was used directly for rigid-body refinement against data from
K378W crystals. The Ca2+-saturated monomeric GCaMP2 structure was
solved using the refined K378W coordinates as a search model. A clear solution
was obtained in space group P21212 with a translation
function Z-score of 39.2 using data between 45.4- and 2.65-Å
resolution. The 8EF-apo GCaMP2 calcium-free mutant structure was solved by
searching for the cpEGFP domain from the Ca2+-dimer structure. A
clear solution was obtained in space group C2 with a translation function
Z-score of 18.9 using data between 31.9- and 2.8-Å resolution.
Subsequently searching for the calcium-free N-terminal or C-terminal lobes of
CaM (PDB code 1CFD (24 (link))) did
not reveal any clear solutions. Some positive difference density was present
in the electron density maps calculated using the cpEGFP domain solution that
suggested the position of the N-terminal lobe of CaM, which was placed
manually into density and refined. The correctness of this CaM N-terminal lobe
placement was indicated by additional positive difference density for the
linker connecting cpEGFP and the CaM N-terminal lobe, which was subsequently
built.
All models were improved by iterative cycles of model building in Coot
(25 ) and positional refinement
in REFMAC (26 ). Final GCaMP2
models have reasonable R-factors and model geometries, as illustrated
in
electron density map for each structure is provided in supplemental Fig.
S1.
| Radiation source | APS 31-ID | ALS BL8.2.2 | APS 31-ID | Copper anode | APS 31-ID |
| Wavelength (Å) | 0.9793 | 1.000 | 0.9793 | 1.5418 | 0.9793 |
| Space group | C2 | P21212 | P41212 | P41212 | C2 |
| Cell dimensions | |||||
| a, b, c (Å) | 126.13, 47.30, 68.94 | 60.49, 68.80, 117.26 | 121.64, 121.64, 97.32 | 120.82, 120.82, 97.35 | 211.87, 47.67, 42.99 |
| α, β, γ (°) | 90, 100.48, 90 | 90, 90, 90 | 90, 90, 90 | 90, 90, 90 | 90, 97.61, 90 |
| Resolution (Å) | 67.79-1.80 (1.90-1.80) | 50.00-2.65 (2.74-2.65) | 25.90-2.00 (2.11-2.00) | 29.30-2.80 (2.90-2.80) | 31.94-2.80 (2.95-2.80) |
| Rsym | 7.6 (42.2) | 7.5 (27.9) | 9.6 (60.1) | 18.4 (57.3) | 15.4 (61.7) |
| I/σI | 18.8 (5.1) | 20.44 (4.2) | 21.5 (4.7) | 8.6 (3.5) | 15.4 (3.2) |
| Completeness (%) | 98.5 (97.6) | 98.7 (92.1) | 100.0 (100.0) | 100.0 (100.0) | 98.9 (98.6) |
| Redundancy | 7.5 (7.6) | 3.1 (3.0) | 14.1 (13.5) | 11.94 (12.04) | 7.1 (7.1) |
| Resolution (Å) | 1.85 | 2.65 | 2.00 | 2.80 | 2.80 |
| Unique reflections | 33,937 | 14,650 | 49,718 | 18,310 | 10,580 |
| Rwork/Rfree | 0.189/0.241 | 0.222/0.280 | 0.194/0.224 | 0.224/0.266 | 0.210/0.283 |
| No. atoms (AU) | 3,333 | 2,799 | 3,472 | 3,182 | 2,430 |
| Protein | 3,097 | 2,767 | 3,185 | 3,154 | 2,425 |
| Ligand/ion | 4 | 3 | 10 | 4 | |
| Water | 198 | 29 | 277 | 24 | 5 |
| B-factors (Å2) | |||||
| Protein | 25.4 | 44.0 | 33.4 | 38.8 | 31.1 |
| Ligand/ion | 19.4 | 75.3 | 38.7 | 41.4 | |
| Water | 32.2 | 29.4 | 37.1 | 23.3 | 12.9 |
| Root mean square deviations | |||||
| Bond lengths (Å) | 0.018 | 0.010 | 0.011 | 0.011 | 0.016 |
| Bond angles (°) | 1.735 | 1.336 | 1.332 | 1.335 | 1.691 |
APS 31-ID, Advanced Photon Source, Beamline 31-ID.
ALS BL8.2.2, Advanced Light Source Beam Line 8.2.2.
Data collected on home source using Rigaku Rotating Copper Anode RUH3R.
B-factors were calculated on the STAN server using MOLEMAN2
(
using a Superdex 200 10/300 GL column (GE Healthcare) at a flow rate of 0.5 ml
min-1 in 20 m
m
5 m
Molecular weights were estimated by comparison with elution volumes of
standard proteins (Bio-Rad).
Sedimentation Velocity Analytical
Ultracentrifugation—Analytical ultracentrifugation of GCaMP2
samples was carried out in a Beckman XL-I analytical ultracentrifuge (Beckman
Coulter, Fullerton, CA) within the Biophysics Instrumentation Facility at the
Massachusetts Institute of Technology. Absorbance scans at 280 nm were
collected on calcium-free (24 μ
μ
AnTi-60 rotor at 42,000 rpm. Data were collected at 20 °C in the same
buffers as the SEC experiments. Absorbance scans were modeled using a
continuous c(s) distribution within Sedfit
(27 (link)), correcting for buffer
density and viscosity and using a partial specific volume of 0.7300
cm3 g-1. Molecular weight of observed species
(
estimated based on the best-fit frictional ratio as determined by Sedfit for
each sample.
amino acid sequence of GCaMP2 illustrating the domain organization. Domains
are colored as depicted in B-D. Carets below the schematic show the
positions of inter-domain linkers whose amino acid sequences are given.
B, stereoview of the structure of the Ca2+-saturated
domain-swapped GCaMP2 dimer, depicted as ribbons. One molecule of the
dimer is colored by domain as in A, the other molecule is colored
light gray. The EGFP chromophore is represented as sticks and calcium ions are shown as orange spheres. C, structure of
Ca2+-saturated GCaMP2 monomer, represented as in B except
the domains are labeled. D, structure of calcium-free GCaMP2,
represented as in B and C. Note that the M13 peptide and the
C-terminal half of CaM are not included in the model due to lack of electron
density, suggesting their flexibility. This and other structure figures were
prepared using PyMOL (Delano Scientific, San Carlos, CA).
GCaMP2—To measure intracellular [GCaMP2], acute brain slices
containing neurons expressing GCaMP2 were prepared and imaged as previously
described (6 (link),
16 (link)). Purified GCaMP2 was
diluted into pipette internal solution supplemented with 1 m
solution was drawn into a thin glass capillary (ID = 0.02 mm, Vitrocom number
RT5002). Their fluorescence intensities were measured under two-photon
excitation with identical parameters (910 nm excitation) to neuron imaging.
0.1 μ
dark current at laser powers used for neuronal imaging.
Intracellular GCaMP2 concentration in neurons with robust fluorescent
responses to action potential firing was estimated by a linear extrapolation
from the purified 10 μ
Intracellular GCaMP2 was assumed to be in the apo state
(6 (link)).
Generation and Screening of GCaMP2 Mutants—Mutants of GCaMP2
were prepared by site-directed mutagenesis (see supplemental Tables S1-S3) and
confirmed by sequencing. Preliminary screening for variants with altered
oligomerization equilibria (supplemental Table S1) was performed by passing
100-μl aliquots of cell-free extract from 200-ml cultures of overexpressed
GCaMP2 over a Superdex 200 10/300 GL column while monitoring the absorption at
280 and 495 nm.
Spectrophotometric Analysis—Absorbance spectra were obtained
in a Safire2 (Tecan) with UVStar 96-well plates (Greiner) for both
the calcium-free (10 m
m
(Greiner) were used. Samples were diluted 10-fold in zero free calcium buffer
(Invitrogen) (30 m
calcium buffer (Invitrogen) (30 m
in 100 m
measurements, samples were dialyzed into 20 m
m
concentration of 10 m
Protein Expression, Purification, and Crystallization—The gene encoding single chain (VH-linker-VL) antibody 80R (scFv) was cloned into pET22b (Novagen) containing an N-terminal periplasmic secretion signal pelB, and a thrombin-removable C-terminal His6 tag. 80R was overexpressed in BL21(DE3) cells at 30 °C for 15 h with 1 m
The gene encoding S1-RBD (residues 318-510) was cloned into vector pAcGP67A (Pharmingen) containing an N-terminal gp67 secretion signal and a thrombin-cleavable C-terminal His6 tag. It was expressed in Sf9 cells (Invitrogen) with a multiplicity of infection = 5 for 72 h. Similar to 80R, S1-RBD was purified from the media with HisBind nickel-nitrilotriacetic acid and Superdex 200 columns, with thrombin digestion. N-Linked glycosylation was removed by incubation with peptide:N-glycosidase F (New England Biolabs) at 23 °C, as monitored by SDS-PAGE. S1 RBD-80R complexes were formed by mixing the two purified components and isolated by gel filtration with Superdex 200 in 10 m
Crystals grew by the hanging drop vapor diffusion method at 17 °C over ∼21 days. For S1-RBD, 2 μl of S1-RBD was mixed with an equal volume of well solution containing 4% w/v polyethylene glycol 4000, 0.1
Data Collection, Structure Determination, and Refinement— X-ray diffraction data were collected at the National Synchrotron Light Source beamline X6A and X29A for S1-RBD crystals, the Stanford Synchrotron Radiation Laboratory beamline 11.1, and at the Advanced Light Source beamlines 5.0.3 and 12.3.1 for crystals of the S1-RBD-80R complex. Glycerol (25%) was used as a cryoprotectant in both cases. All the data were processed with DENZO and SCALEPACK or with the HKL2000 package (8 ). Crystals of S1 RBD adopt space group P43212 with unit cell dimensions a = 75.9 and c = 235.8 (
Data collection and refinement statistics
| Cell parameters | a = 75.9, c = 235.9 Å | a = 47.5, b = 175.9, c = 67.6 Å; β = 96.6° |
| Space group | P43212 | P21 |
| Resolution (Å) | 2.2 | 2.3 |
| Total reflections | 233011 | 159047 |
| Unique reflections | 36036 | 51915 |
| Completeness (%) | 99.9 (99.9) | 93.8 (87.0) |
| Average I/σ(I) | 24.7 (2.0) | 8.8 (1.9) |
| Rmerge | 0.098 (0.739) | 0.145 (0.571) |
| Redundancy | 6.5 | 3.1 |
| Rwork | 0.182 (0.230) | 0.248 (0.301) |
| Rfree (5% data) | 0.213 (0.289) | 0.295 (0.391) |
| r.m.s.d. bond distance (Å) | 0.013 | 0.009 |
| r.m.s.d. bond angle (°) | 1.49 | 1.22 |
| Average B value | 50.0 | 37.1 |
| Solvent atoms | 152 | 470 |
| Residues in most favored regions | 276 | 631 |
| Residues in additional allowed regions | 35 | 81 |
| Residues in generously allowed regions | 3 | 5 |
| Residues in disallowed regions | 0 | 0 |
Numbers in parentheses correspond to the highest resolution shell (2.28-2.20 Å for S1 RBD; 2.29-2.38 Å for S1 RBD-80R)
Numbers in parentheses correspond to the highest resolution shell (2.26-2.20 Å for S1 RBD; 2.29-2.38 Å for S1 RBD-80R)
r.m.s.d., root meant square deviation
where A and B were obtained by least square fitting of the averaged measured intensities. The ratio of the parameters B and A (B/A = 0.65) coincided with the height ratio of the Patterson peak at (1/3, 0, 0), as required by the lattice-translocation theory presented by Wang. The corrected intensity distribution (
The structure of uncomplexed S1-RBD (which showed no lattice defects) was determined by molecular replacement with PHASER (13 (link)) using S1-RBD from the structure of the S1-RBD-ACE2 complex (Protein Data Bank code 2AJF) as the search model. The asymmetric unit contains two molecules of S1-RBD arranged as a symmetric dimer. The final model includes residues 320-503 of both monomers and 152 water molecules.
Geometric parameters are excellent as assessed with PRO-CHECK (18 ) (
The cytoarchitecture of the PFC was studied in ten adult, male mice (strain C57BL/6) of similar weight (approximately 20 g). These control mouse brains were kindly donated and immersion fixed by Dr. H. Manji, NIMH, USA. All animal procedures were in strict accordance with the NIH animal care guidelines. The histological processing of these brains was performed at the laboratory of Dr. Rajkowska. The brains were embedded in 12% celloidin, cut into 40-μm serial sections using a sliding microtome and Nissl (1% cresyl violet) stained. Celloidin was chosen as an embedding medium to allow for the preparation of ‘thick’ sections with clear morphology and high contrast of Nissl-stained neurons and glial cells. In these immersion-fixed brains, any spots showing pycnotic reaction were not incorporated in this study.
In addition to these ten mice, four adult male mice (C57BL/6 strain) were stained for dopamine and four adult male mice for AChE, myelin, and immunohistochemically for SMI, PV and CB. For each staining, a different set of sections with several consecutive sections stained with Nissl at HBMU’s laboratory was used. The antibodies applied were the dopamine (DA) antibody (Geffard et al. 1984 (link)), SMI-32 antibody (Sternberger Monoclonals Inc., Baltimore, MD, USA: monoclonal antibody to one epitope of non-phosphorylated tau neurofilaments, lot number 11), SMI-311antibody (pan-neuronal neurofilament marker cocktail of several monoclonal antibodies for several epitopes of non-phosphorylated tau protein, Sternberger Monoclonals Inc., Baltimore, MD, USA: lot number 9) (SMI antibodies are presently distributed through Covance Research Products, USA), monoclonal anti-CB D-28K antibody (Sigma, St. Louis, MO, USA: product number C-9848, clone number CB-955, lot number 015K4826), and monoclonal anti-PV antibody (Sigma, St. Louis, MO, USA: product number P-3171, clone number PA-235, lot number 026H4824). Mice to be stained for DA were intracardially perfused under deep pentobarbital anesthesia (1 ml/kg body weight, i.p.), with saline followed by fixative. For DA staining, the fixative was 5% glutaraldehyde in 0.05 M acetate buffer at pH 4.0. After perfusion, the brains were immersed in 0.05 Tris containing 1% sodium disulfite (Na2S2O5) at pH 7.2 (De Brabander et al. 1992 (link)). Mouse PFC was sectioned at 40 μm by a vibratome. These sections were stained overnight in a cold room at 4°C using the polyclonal primary antibody sensitive to DA that was raised in the Netherlands Institute for Brain Research (NIBR) (Geffard et al. 1984 (link)), the specificity of which had been demonstrated previously (Kalsbeek et al. 1990 (link)). DA antiserum was diluted 1:2,000 in 0.05 M Tris containing 1% Na2S2O5 and 0.5% Triton X-100, pH 7.2. After overnight incubation, the sections were washed three times with Tris-buffered saline (TBS) and subsequently incubated in the secondary antibody goat–antirabbit, also raised in NIBR at 1:100 for 1 h. After having been rinsed 3× in TBS, it was incubated in the tertiary antibody, peroxidase–antiperoxidase, at 1:1,000 for 60 min. Both the secondary and the tertiary antibodies were diluted in TBS with 0.5% gelatine and 0.5% Triton X-100. For visualization, the sections were transferred into 0.05% diaminobenzidine (DAB; Sigma) with 0.5% nickel ammonium sulfate. The reaction was stopped after a few minutes by transferring the sections to TBS (3 × 10 min), then the sections were mounted on slides, air dried, washed, dehydrated and coverslipped.
Mice to be stained with anti-PV, anti-CB and SMI-32 and SMI-311 were fixed with 4% formaldehyde solution in 0.1 M phosphate buffer at pH 7.6. Mouse PFC was sectioned at 40 μm by a vibratome. To prevent endogenous peroxidase activity, free-floating sections were pretreated for 30 min in a Tris-buffered saline (TBS) solution containing 3% hydrogen peroxide and 0.2% Triton X-100. To prevent non-specific antibody staining, these sections were placed in a milk solution (TBS containing 5% nonfat dry milk and 0.2% Triton X-100) for 1 h. Incubation of the primary antibody, directly after the milk step was carried out overnight in a cold room at 4°C. The primary antibodies were diluted in the above-mentioned milk solution: SMI-32 and SMI-311 at 1:1,000, PV antibody at 1:1,000, and CB antibody at 1:250. For the monoclonal SMI-32, SMI-311, PV and CB antibodies, raised in mice, we used peroxidase-conjugated rabbit–antimouse (1:100 in 5% milk solution with 0.2% Triton X-100) as a secondary antibody. Visualization took place in 0.05% diaminobenzidine enhanced with 0.2% nickel ammonium sulfate. The reaction was stopped after a few minutes by transferring these sections to TBS (3 × 10 min), after which the sections were rinsed in distilled water, mounted on slides, air dried, washed, dehydrated and coverslipped. Control sections that were incubated according to the same procedure as described above, omitting the primary antibody, were all negative. All sections were cut coronally, because the coronal plane offers in general the best view to differentiate between the subareas of the rodent PFC (Uylings et al. 2003 (link); Van de Werd and Uylings 2008 (link)).
Sections were processed for AChE staining according to the protocol described by Cavada et al. (1995 (link)). The sections were incubated overnight in a solution of cupric sulfate and acetate buffer at pH 5 to which acetylthiocholine iodide and ethopropazine were added just before the start of incubation. After rinsing, the sections were developed in a sodium sulfide solution until a light brown color appeared and subsequently intensified to a dark brown color in a silver nitrate solution. Finally, the sections were differentiated after rinsing in a thiosulfate solution, dehydrated and mounted. In all steps, the solutions and sections were shaken constantly. The myelin was stained with silver by physical development according to Gallyas (1979 (link)). The sections were first placed in 100% ethanol and then immersed in a 2:1 solution of pyridine and acetic acid for 30 min. After rinsing, they were placed in an ammonium silver nitrate solution and after rinsing with 0.5% acetic acid, the sections were immersed in the optimal physical developer solution at room temperature (Gallyas 1979 (link)) until they showed good stain intensity under the microscope. Then the development of the staining was stopped in 0.5% acetic acid and the sections were dehydrated and mounted with Histomount. The sections were studied at intervals of 80–160 μm, and examined under a light microscope at a 63× magnification.
In addition to these ten mice, four adult male mice (C57BL/6 strain) were stained for dopamine and four adult male mice for AChE, myelin, and immunohistochemically for SMI, PV and CB. For each staining, a different set of sections with several consecutive sections stained with Nissl at HBMU’s laboratory was used. The antibodies applied were the dopamine (DA) antibody (Geffard et al. 1984 (link)), SMI-32 antibody (Sternberger Monoclonals Inc., Baltimore, MD, USA: monoclonal antibody to one epitope of non-phosphorylated tau neurofilaments, lot number 11), SMI-311antibody (pan-neuronal neurofilament marker cocktail of several monoclonal antibodies for several epitopes of non-phosphorylated tau protein, Sternberger Monoclonals Inc., Baltimore, MD, USA: lot number 9) (SMI antibodies are presently distributed through Covance Research Products, USA), monoclonal anti-CB D-28K antibody (Sigma, St. Louis, MO, USA: product number C-9848, clone number CB-955, lot number 015K4826), and monoclonal anti-PV antibody (Sigma, St. Louis, MO, USA: product number P-3171, clone number PA-235, lot number 026H4824). Mice to be stained for DA were intracardially perfused under deep pentobarbital anesthesia (1 ml/kg body weight, i.p.), with saline followed by fixative. For DA staining, the fixative was 5% glutaraldehyde in 0.05 M acetate buffer at pH 4.0. After perfusion, the brains were immersed in 0.05 Tris containing 1% sodium disulfite (Na2S2O5) at pH 7.2 (De Brabander et al. 1992 (link)). Mouse PFC was sectioned at 40 μm by a vibratome. These sections were stained overnight in a cold room at 4°C using the polyclonal primary antibody sensitive to DA that was raised in the Netherlands Institute for Brain Research (NIBR) (Geffard et al. 1984 (link)), the specificity of which had been demonstrated previously (Kalsbeek et al. 1990 (link)). DA antiserum was diluted 1:2,000 in 0.05 M Tris containing 1% Na2S2O5 and 0.5% Triton X-100, pH 7.2. After overnight incubation, the sections were washed three times with Tris-buffered saline (TBS) and subsequently incubated in the secondary antibody goat–antirabbit, also raised in NIBR at 1:100 for 1 h. After having been rinsed 3× in TBS, it was incubated in the tertiary antibody, peroxidase–antiperoxidase, at 1:1,000 for 60 min. Both the secondary and the tertiary antibodies were diluted in TBS with 0.5% gelatine and 0.5% Triton X-100. For visualization, the sections were transferred into 0.05% diaminobenzidine (DAB; Sigma) with 0.5% nickel ammonium sulfate. The reaction was stopped after a few minutes by transferring the sections to TBS (3 × 10 min), then the sections were mounted on slides, air dried, washed, dehydrated and coverslipped.
Mice to be stained with anti-PV, anti-CB and SMI-32 and SMI-311 were fixed with 4% formaldehyde solution in 0.1 M phosphate buffer at pH 7.6. Mouse PFC was sectioned at 40 μm by a vibratome. To prevent endogenous peroxidase activity, free-floating sections were pretreated for 30 min in a Tris-buffered saline (TBS) solution containing 3% hydrogen peroxide and 0.2% Triton X-100. To prevent non-specific antibody staining, these sections were placed in a milk solution (TBS containing 5% nonfat dry milk and 0.2% Triton X-100) for 1 h. Incubation of the primary antibody, directly after the milk step was carried out overnight in a cold room at 4°C. The primary antibodies were diluted in the above-mentioned milk solution: SMI-32 and SMI-311 at 1:1,000, PV antibody at 1:1,000, and CB antibody at 1:250. For the monoclonal SMI-32, SMI-311, PV and CB antibodies, raised in mice, we used peroxidase-conjugated rabbit–antimouse (1:100 in 5% milk solution with 0.2% Triton X-100) as a secondary antibody. Visualization took place in 0.05% diaminobenzidine enhanced with 0.2% nickel ammonium sulfate. The reaction was stopped after a few minutes by transferring these sections to TBS (3 × 10 min), after which the sections were rinsed in distilled water, mounted on slides, air dried, washed, dehydrated and coverslipped. Control sections that were incubated according to the same procedure as described above, omitting the primary antibody, were all negative. All sections were cut coronally, because the coronal plane offers in general the best view to differentiate between the subareas of the rodent PFC (Uylings et al. 2003 (link); Van de Werd and Uylings 2008 (link)).
Sections were processed for AChE staining according to the protocol described by Cavada et al. (1995 (link)). The sections were incubated overnight in a solution of cupric sulfate and acetate buffer at pH 5 to which acetylthiocholine iodide and ethopropazine were added just before the start of incubation. After rinsing, the sections were developed in a sodium sulfide solution until a light brown color appeared and subsequently intensified to a dark brown color in a silver nitrate solution. Finally, the sections were differentiated after rinsing in a thiosulfate solution, dehydrated and mounted. In all steps, the solutions and sections were shaken constantly. The myelin was stained with silver by physical development according to Gallyas (1979 (link)). The sections were first placed in 100% ethanol and then immersed in a 2:1 solution of pyridine and acetic acid for 30 min. After rinsing, they were placed in an ammonium silver nitrate solution and after rinsing with 0.5% acetic acid, the sections were immersed in the optimal physical developer solution at room temperature (Gallyas 1979 (link)) until they showed good stain intensity under the microscope. Then the development of the staining was stopped in 0.5% acetic acid and the sections were dehydrated and mounted with Histomount. The sections were studied at intervals of 80–160 μm, and examined under a light microscope at a 63× magnification.
Most recents protocols related to «Nickel sulfate»
Example 1
In this example, granulated/atomized FeNi particles are provided directly to a POX autoclave for a copper assisted POX leach; leaching and hematite production occur in one unit operation.
Example 2
The process of Example 1 may be carried out with the following modification: instead of the FeNi alloy being treated directly with a pressure oxidation (POX) leach, the FeNi alloy may instead be treated with a leach in a vessel (such as a column) in the presence of peroxide and sulfuric acid.
As an alternative to or a supplement to the copper assisted POX leach described in Example 1 and
The recycled ratio of the PLS may be adjusted to reduce the formation of basic sulphates or goethite. The desirable iron product in the process is hematite due to its high iron content, low sulphur content and marketability potential.
Following the oxidizing leach, the examples can be carried out in the same way, including solids removal and then copper cementation (if copper was added to the oxidizing leach). Further processing steps may include those listed above for Example 1.
Example 3
The process of Example 1 may be carried out with the following modification: instead of the FeNi alloy being treated directly with a pressure oxidation (POX) leach, the FeNi alloy may instead be treated with a leach in a vessel (such as a column) in the presence of added copper.
As an alternative to or a supplement to the copper assisted POX leach described in Example 1 and
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This research utilized analytical-grade chemicals and distilled water solution preparations. Tetraamminecopper(II) sulfate monohydrate ([Cu(NH3)4]SO4·H2O) was procured from Sigma-Aldrich (St. Louis, MO, USA). A 500 mg/L Ni(II) stock solution was prepared by dissolving nickel dichloride (NiCl2) in distilled water, and working solutions were subsequently diluted from this stock. The pH value was adjusted using 0.1 M hydrochloric acid (HCl) and 0.1 M sodium hydroxide (NaOH). Sodium chloride (NaCl) was employed to adjust the ionic strengths. Phragmites australis was harvested from Baiyun Lake (117.40 °E, 36.86 °N, Shandong Province, China) and washed five times with distilled water. Then, Phragmites australis was dried at 105 °C for 48 h to a constant weight and then comminuted into fragments (0.45–1.0 mm).
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All of the reagents and materials were noted to be of analytical quality. Vancomycin (≥85%, CAS no. 1404-93-9), tetra-chloroauric acid trihydrate (HAuCl4.3H2O; 99.9%, CAS no. 16961-25-4), polyethyleneimine (50% w/v in H2O; Mw. 750k, CAS no. 9002-98-6), glutathione, BSA (Bovine serum albumin) and formaldehyde (36.0% in H2O) were obtained from Sigma Aldrich (Mumbai, India). Nickel sulfate, mercuric chloride (99.5%), potassium chloride (99%), Nickel sulfate, magnesium chloride, sodium chloride (99%), ammonium chloride, tryptophan, tyrosine, and solvents were purchased from Merck Life Sciences (Bangalore, India). The other plasticware and glassware were acquired from Tarson (Mumbai, India).
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Sodium hydroxide is a chemical compound with the formula NaOH. It is a white, odorless, crystalline solid that is highly soluble in water and is a strong base. It is commonly used in various laboratory applications as a reagent.
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3,3'-diaminobenzidine is a chemical compound commonly used as a chromogenic substrate in various laboratory techniques, such as immunohistochemistry and enzyme-linked immunosorbent assays (ELISA). It is a sensitive and specific reagent that can be used to detect the presence of target proteins or enzymes in biological samples.
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Nickel(II) sulfate hexahydrate is a chemical compound with the formula NiSO4·6H2O. It is a green crystalline solid that is soluble in water. The compound is commonly used as a starting material in the production of other nickel compounds and in various industrial and laboratory applications.
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Nickel sulfate is a chemical compound with the formula NiSO4. It is a crystalline solid that is soluble in water. Nickel sulfate is commonly used as a source of nickel ions in various industrial and laboratory applications.
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NaCl is a chemical compound commonly known as sodium chloride. It is a white, crystalline solid that is widely used in various industries, including pharmaceutical and laboratory settings. NaCl's core function is to serve as a basic, inorganic salt that can be used for a variety of applications in the lab environment.
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Sodium sulfate is a chemical compound with the formula Na₂SO₄. It is a white, crystalline solid that is commonly used as a desiccant, a filler in detergents, and in the production of glass, paper, and textiles.
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Sodium dodecyl sulfate (SDS) is a commonly used anionic detergent for various laboratory applications. It is a white, crystalline powder that has the ability to denature proteins by disrupting non-covalent bonds. SDS is widely used in biochemical and molecular biology techniques, such as protein electrophoresis, Western blotting, and cell lysis.
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Nickel sulfate hexahydrate is an inorganic compound with the chemical formula NiSO4·6H2O. It is a green crystalline solid that is soluble in water. Nickel sulfate hexahydrate is commonly used in various industrial and research applications that require a source of nickel ions.
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The Vectastain Elite ABC kit is a specialized laboratory equipment used for the detection and visualization of target proteins or antigens in biological samples. It utilizes an avidin-biotin complex (ABC) system to amplify the signal, enabling researchers to achieve high sensitivity and consistent results in their immunohistochemical or immunocytochemical analyses.
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Sodium hydroxide is a chemical compound with the formula NaOH. It is a white, crystalline solid that is highly soluble in water. Sodium hydroxide has a wide range of applications in various industries, including as a pH regulator, cleaning agent, and chemical intermediate.