Hsp104 was purified as previously described (DeSantis et al., 2014 (link)). In brief, Hsp104 was expressed in BL21 (DE3) RIL cells, lysed via sonication in lysis buffer [50 mM Tris-HCl pH = 8.0, 10 mM MgCl2, 2.5% glycerol, 2 mM β-mercaptoethanol, 2.5 µM PepstatinA, and cOmplete Protease Inhibitor Cocktail (one mini EDTA-free tablet/50 mL, Millipore Sigma)], clarified via centrifugation at 30,597xg and 4°C for 20 min, and purified on Affi-Gel Blue Gel (Bio-Rad). Hsp104 was eluted in elution buffer (50 mM Tris-HCl pH = 8.0, 1M KCl, 10 mM MgCl2, 2.5% glycerol, and 2 mM β-mercaptoethanol) and then exchanged into storage buffer (40 mM HEPES-KOH pH = 7.4, 500 mM KCl, 20 mM MgCl2, 10% glycerol, 1 mM DTT). The protein was diluted to 10% in buffer Q (20 mM Tris-HCl pH = 8.0, 50 mM NaCl, 5 mM MgCl2, and 0.5 mM EDTA) and loaded onto a 5 mL RESOURCE Q anion exchange chromatography (GE Healthcare). Hsp104 was eluted via linear gradient of buffer Q+ (20 mM Tris pH = 8.0, 1M NaCl, 5 mM MgCl2, and 0.5 mM EDTA). The protein was then exchanged into storage buffer and snap frozen. Protein purity was determined to be >95% by SDS-PAGE and Coomassie staining.
Affi gel blue gel
Manufactured by Bio-Rad
Sourced in United States
The Affi-Gel Blue Gel is a chromatographic medium designed for the purification of proteins. It is a cross-linked agarose gel that contains Cibacron Blue F3GA, a dye ligand, which can interact with a variety of proteins. The gel is used for the selective adsorption and separation of proteins based on their affinity for the dye ligand.
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Lab products found in correlation
Resource q anion exchange chromatography, by GE Healthcare (2 mentions)
Complete protease inhibitor cocktail, by Merck Group (2 mentions)
Pepstatin a, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Superdex 75 hr 10 30 column, by GE Healthcare (1 mentions)
Deae cellulose, by Merck Group (1 mentions)
Sp sepharose, by GE Healthcare (1 mentions)
Recombinant human tgf β1 protein, by R&D Systems (1 mentions)
Cell culture insert, by BD (1 mentions)
14 protocols using affi gel blue gel
1
Hsp104 Protein Purification Protocol
2
Zebrafish Kidney Explant Culture
3
Purification of Trypsin Inhibitor from Gold Beans
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A water extract of gold beans Phaseolus vulgaris cv. “gold bean” from Mainland China (260 g) was made by homogenizing them in distilled water (6 mL/g). The homogenate was then centrifuged (14,000× g for 25 min at 4 °C). The supernatant was collected and loaded on a 5 × 20 cm column of DEAE-cellulose (Sigma, St. Louis, MI, USA) in 10 mM Tris-HCl buffer (pH 7.4). Following removal of unadsorbed proteins (fraction D1), the column was eluted sequentially with 0.2 M NaCl and 1 M NaCl in the Tris-HCl buffer. Fraction D2 eluted with 0.2 M NaCl was dialyzed and then chromatographed on a 5 × 15 cm column of Affi-gel blue gel (Bio-Rad, Woodinville, WA, USA) in 10 mM Tris-HCl buffer (pH 7.4). The unadsorbed proteins (fraction B1) were dialyzed against 10 mM NH4Ac buffer (pH 5) and applied on a 2.5 × 20 cm column of SP-sepharose (GE Healthcare, Uppsala, Sweden). After elution of unadsorbed proteins (fraction S1), the column was eluted with a 0–1 M NaCl concentration gradient in the NH4Ac buffer. The first adsorbed fraction (S2) was then subjected to gel filtration on a Superdex 75 HR 10/30 column (GE Healthcare) in 0.2 M NH4HCO3 buffer (pH 8.5). The second absorbance peak (SU2) represented purified trypsin inhibitor (GBTI).
4
Mandibular Arch Explant Culture Assay
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The mandibular arch was dissected from E11.5 embryos and placed oral-epithelium-side up onto MCE membrane filter 0.8 µm (Millipore #AAWP04700) supported by Millicell cell culture inserts (0.4 µm, 30 mm diameter; Millipore #PICM0RG50) floating atop BGJb culture medium (Gibco #12591038) with the addition of 100 µg/ml ascorbic acid in 6-well tissue culture plates. Culture media was carefully applied to cover the explants such that the tissue sat at the air-media interface. Agarose beads (Affi-Gel Blue Gel, Bio-Rad #153-7302) were washed in PBS, then all liquid removed and beads air-dried slightly, before addition of 10% BSA or recombinant human IGF1 100 µg/ml (R&D Systems #291-G1), and incubated for at least 30 min at room temperature. Beads were implanted into the mandible tissue at time of explant. Explants were cultured up to 6 days, with exchange of fresh culture medium every second day. For Alcian Blue staining, explants were fixed on the membrane in 95% ethanol, and incubated in staining solution (0.02% Alcian Blue, 5% glacial acetic acid in 70% ethanol) for 2 days, then cleared in 1% KOH for several hours.
5
Beads Implantation for Tgfβ1 Delivery
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Beads (Affi-Gel Blue Gel; Bio-Rad) soaked in either recombinant human Tgfβ1 protein (R&D Systems) or control buffer were implanted into the snouts of juvenile (2.9–5.3 cm SL) LT (n = 9 Tgfβ1-treated, n = 9 control), TRC (n = 5 Tgfβ1-treated, n = 3 control), and TT (n = 6 Tgfβ1-treated, n = 5 control). Because of the nature of this experiment, we used larger juvenile fish, but they were still in the range of those used for the histological analysis. A small incision was made in anesthetized fish parallel to the snout, and a path was bored using a sewing needle to the tip of the snout. Four incubated beads were then pushed through the incision and guided to the tip of the snout, taking care to leave tissue as undisturbed as possible. All fish were recovered from the surgery and were collected either 12 h later for gene expression analysis or 7 d later for morphological analysis.
6
Bmp7 Bead Implantation in BPs
7
Bmp7 Bead Implantation in BPs
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BPs were dissected and cultured as described. Agarose beads (Affi-Gel® Blue gel, Bio-Rad) were incubated for 1 hour at room temperature in either bovine serum albumin (BSA) (SIGMA Aldrich) or BSA with 0.5 μg/μL of Bmp7 protein. Treated beads were subsequently placed at the proximal end of the BP using fine forceps. Cultures were then maintained for 6 days after which they were fixed in 4% PFA and processed for immunohistochemistry.
8
Cdc42 Knockout Impacts Embryo Development
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Cdc42f/f mice containing loxP sites flanking exon 2 of the Cdc42 gene were produced by homologous recombination as described previously [45 (link)]. PgrCre/+ mouse models were utilized to delete Cdc42 in the female reproductive tract [23 ]. The conditional knockout females (PgrCre/+Cdc42f/f) and Cdc42f/f control littermates were used in the experiments. Female mice at least 8 weeks old were mated with fertile WT males to induce pregnancy (vaginal plug = day 1 of pregnancy). The number of embryos from uterus and oviduct was calculated as a percentage of the total morula and blastocyst stage embryos collected. Mice that failed to recover any embryos were excluded from the statistical analysis. Mouse blood samples were collected on day 4 in the morning and serum P4 as well as E2 levels were measured by radioimmunoassay. Affi-Gel Blue Gel (Bio-Rad; 100‒200 mesh, no.153-7302) beads about the size of an eight-cell embryo were transferred into the oviduct umbrella of D1 pseudopregnant mice, and mice were sacrificed on D4 to check the localization of transferred blue beads. All mice were housed in the Animal Care Facility of Xiamen University, in accordance with the guidelines for the care and use of laboratory animals.
9
Netrin-1 Bead Implantation in Brain Slices
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We cut 200–250-μm thick sections of E16.5 Unc5C or EGFP electroporated brains using a vibratome and cultured them on cell culture inserts (1-ct pore size, BD Biosciences) according to the protocol described previously41 (link)42 (link). The slices were cultured in a medium consisting of complete Hank's Balanced Salt Solution, basal medium Eagle, 20 mM D -glucose, 1 mM L -glutamine, penicillin (100 U ml−1), streptomycin (0.1 mg ml−l), N2 supplement (100 μ, per 12.5 ml) and 10% heat-inactivated horse serum (vol/vol). Agarose beads (Affi-gel Blue gel, Bio-Rad Laboratories) were washed three times with sterile water and four times with PBS. For coating the beads with Netrin1, 20 μl of bead solution was mixed with 10 μl of a Netrin1 solution (250 ng μg−1 R&D Biosciences) and incubated overnight at 4 °C. BSA-coated beads were used as a control for which 20 μl of bead solution was mixed with 20 μl of BSA (1 mg ml−l). Before placing the beads on the slices, they were washed with PBS and then resuspended in 100 μl. of slice culture medium. Using a mouth pipette, the beads of the desired size were picked and placed over the midline in the region where the CC crosses. The cultured slices were immunostained (see above) after 3 days.
10
Albumin Depletion from Serum
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Albumin removal was performed using Affi-Gel blue gel, 50–100 mesh (Bio-Rad) according to the manufacturer's protocol. Lipoprotein-depleted serum collected as described above was dialyzed against buffer A, and 100 mg of dialyzed sample was loaded onto the 10 ml Affi-Gel blue gel column equilibrated with buffer A. Its effluent recovered by the wash with 8 ml buffer A was used as the effluent fraction.
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