Surebeads protein a magnetic beads

The growth factor EGF was from R&D Systems (Minneapolis, MN, USA). TPA and bafilomycin A1 were purchased from Sigma-Aldrich. Rapamycin and MG132 were purchased from Calbiochem (San Diego, CA, USA). 3-metyladenine was the product of Adipo Gen Life Sciences (San Diego, CA, USA). Protein assay kits and Sure Beads Protein A Magnetic Beads were obtained from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). Magnetic Racks were purchased from Invitrogen (Waltham, MA, USA). Protease Inhibitor Cocktail Tablets (Complete Mini) were purchased from Roche Diagnostic GmbH (Mannheim, Germany). RNAiso Plus was obtained from Takara (Kusatsu, Japan), High Capacity cDNA Reverse Transcription Kit and Power Up SYBR Green Master Mix were the products of Thermo Fisher Scientific (Waltham, MA, USA).

The binding of Hsp60 WT/GW/AD(Cys) and α-synuclein was detected by immunoprecipitation combined with Western blot analysis. Hsp60 WT/GW/AD(Cys) and α-synuclein were agitated for 1 h before immunoprecipitation, and the mixture was used as the sample. The SureBeads™ Protein A Magnetic Beads (Bio-Rad, Hercules, CA, USA) were washed with PBS-T (Phosphate-Buffered Saline (Thermo Fisher Scientific, Waltham, MA, USA) + 0.1% Tween) three times, followed by addition of 200 μL PBS-T and 2 μL antibody (anti-Hsp60 (Mab11-13, mouse, abcam, Cambridge, UK for Hsp60 WT/GW detection), anti-6×His Tag (HIS.H8, mouse, Thermo Fisher Scientific, Waltham, MA, USA for AD(Cys) detection), or anti-α-synuclein (MJFR1, rabbit, abcam, Cambridge, UK)). After rotation for 10 min at 4 °C, the beads were magnetically retained, and the supernatant was discarded. The samples were added to the beads with bound antibodies and incubated with rotation for 4 h at 4 °C. The beads were again magnetically retained, and the supernatant was discarded. Then the beads were washed with PBS-T three times. Proteins were released from the beads by adding 2×SDS loading buffer and boiling for 3 min.

HEK293T cells were transfected with expression vectors for A3B-HA or its mutants with or without expression vectors for FLAG-PKACA using the XtremeGENE HP DNA Transfection Reagent (Roche). At 36 to 48 hours after transfection, cells were lysed with radioimmunoprecipitation (RIPA) buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% (v/v) Triton X-100, 0.1% (v/v) SDS, 0.1% (v/v) sodium deoxycholate, complete Protease Inhibitor Cocktail (Roche), PhosSTOP (Roche)) and immunoprecipitated using the Anti-HA (12CA5) antibody (Roche) or the anti-FLAG (M2) antibody (F3165 Sigma-Aldrich) along with SureBeads Protein A Magnetic Beads (Bio-Rad) at 4 °C, followed by immunoblotting with anti-HA (12CA5) Ab or anti-FLAG (M2) antibodies.

hFasLECD containing single deletion mutation from 103 to 138 and double substitution mutations (N184Q and N250Q) [hFasLECD (139-281, N184Q, N250Q)] with an N-terminal FLAG-(GlyCysGlyGlyGlyGly) tag sequence (NFG1CG4-hFasLECD) and that with an N-terminal FLAG-(Gly)₅ tag sequence (NFG5-hFasLECD) were prepared as described (Muraki 2008 (link), 2014b (link)). FL-5Mal was obtained from Tokyo Chemical Ind. Dimethyl Sulfoxide, Super Dehydrated (Dry DMSO), l-Cysteine hydrochloride monohydrate and pre-cast gels for SDS-PAGE analysis (Supersep Ace, 10–20 % gradient gels) were from Wako Pure Chemicals Ind. BCA protein assay kit and Tris-(2-carboxyethyl)phosphine (TCEP) neutral pH solution were purchased from Thermo Fisher Scientific. Ion-exchange chromatography and size-exclusion chromatography were performed using prepacked columns from GE healthcare. Ultracentrifugation devices for sample concentration [Amicon Ultra 4 and 15, molecular-weight cut off (MWCO): 10 kDa] were supplied from Merck Millipore. Phosphate, acetate and Tris-hydrochloride buffers were the products of Nakarai Tesque (the former two) and Nippon Gene, respectively. SureBeads Protein A Magnetic Beads and washing buffer reagents used in immunoprecipitation experiments were from Bio-Rad Laboratories and Roche Diagnostics. Chemical structure of FL-5Mal was drawn using ChemBioDraw Ultra, ver. 14.

Cells were harvested and lysed with Pierce IP Lysis Buffer (Thermo Fisher Scientific, Waltham, MA, USA) plus s complete protease inhibitor cocktail (Roche, Basel, Switzerland). Surebeads Protein A Magnetic Beads (Bio-Rad Laboratories, Hercules, CA, USA) were incubated with indicated antibodies for 10 min at room temperature. The beads were added to samples and incubated overnight at 4 °C. Immunoprecipitates were washed and eluted in SDS sample buffer for subsequent immunoblot analysis.

The protocol followed the manual of SureBeads™ Protein A Magnetic Beads (Bio-Rad, Cat#1614013). In brief, 5 μg mouse anti-LPS antibody was incubated with 100 μl protein A magnetic beads at 4°C for 1 h, and then the beads were washed with 1 ml Phosphate buffered saline with 0.1% Tween® 20 Detergent (PBST) three times. After washing, 70 μg recombinant Der m 2 with or without 100 μg LPS was added and incubated overnight at 4°C on the rotating mixer. After incubation, the mixed solutions were removed and washed with PBST three times. Finally, 20 mM glycine pH 2.0 was added to elute the proteins, and then the elution was transferred to 1.5 M Tris-HCl pH 8.0 to equilibrate the pH value. The eluted protein solutions were further analyzed by Western blot with the anti-Der m 2 antibody and the rabbit anti-mouse HRP antibody (Abcam, Cat# ab97046).