Plasma-derived human C3 and C3b were purchased from Complement Technology, Inc., Tyler, TX (and stored at −80 °C). Native C3 was depleted of low amounts of contaminating C3(H2O) using cation-exchange chromatography (34 (link)). C3(H2O) (i.e. C3(N) in this case but identical to C3(H2O)) was prepared by incubating C3 at 37 °C with 200 mm methylamine (CH3NH2) at pH 8.3 for three hours. The C3(H2O) was then isolated from any other intermediates using cation-exchange chromatography (34 (link)). Chromatography in both cases was performed using a Mini S PC 3.2/3 column (GE Healthcare, Little Chalfont, UK) at a flow-rate of 500 μl/min at 4 °C and a gradient from 0 to 325 mm NaCl. Immediately after purification, C3, C3(H2O) and C3b samples were exchanged, using 30-kDa molecular weight cutoff (MWCO) filters (Millipore, Cork, Ireland), into cross-linking buffer (20 mm HEPES-KOH, pH 7.8, 20 mm NaCl and 5 mm MgCl2) with a final concentration of 2 μm . C3, C3b and C3(H2O) samples were prepared in two separated batched and used for “experiment I” and “experiment II” respectively.
Mwco filter
Manufactured by Merck Group
Sourced in Ireland, United States, Australia
MWCO filters are membrane-based separation devices used to separate molecules based on their molecular weight. They function by allowing the passage of smaller molecules while retaining larger ones, enabling the purification or fractionation of solutions.
Automatically generated - may contain errors
Lab products found in correlation
Flag m2 magnetic beads, by Merck Group (2 mentions)
3xflag peptide, by Merck Group (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Superdex 200 hr10 300, by GE Healthcare (1 mentions)
Hek293t 17, by American Type Culture Collection (1 mentions)
Accuri flow cytometer, by BD (1 mentions)
Pspax2, by Addgene (1 mentions)
Pmd2 g, by Addgene (1 mentions)
French press, by Thermo Fisher Scientific (1 mentions)
Type 50.2 ti rotor, by Beckman Coulter (1 mentions)
Sw28 rotor, by Beckman Coulter (1 mentions)
Wheat germ agglutinin (wga), by Vector Laboratories (1 mentions)
Jacalin, by Vector Laboratories (1 mentions)
Concanavalin a (cona), by Vector Laboratories (1 mentions)
Mops running buffer, by Thermo Fisher Scientific (1 mentions)
Colloidal blue staining kit, by Thermo Fisher Scientific (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Pbs blocking buffer, by LI COR (1 mentions)
Donkey anti rabbit alexa fluor 790, by Jackson ImmunoResearch (1 mentions)
Mouse anti actin antibody, by Merck Group (1 mentions)
28 protocols using mwco filter
1
Purification and Preparation of C3 Variants
2
Cross-linking and MS-based Protein Analysis
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Proteins were cross-linked separately
using a 1:1 weight-to-weight (w/w) cross-linker to protein ratio.
C3b buffer was exchanged using 30 kDa molecular weight cutoff (MWCO)
filters (Millipore, Cork, Ireland) into cross-linking buffer (20 mM
HEPES, 20 mM NaCl, 5 mM MgCl2, pH 7.8). HSA, creatine kinase, myoglobin,
ovotransferrin, catalase, and cytochrome C were dissolved in cross-linking
buffer. Protein and cross-linker were mixed to a final concentration
of 1 mg/mL each. The mixture was incubated on ice for 2 h before the
reaction was stopped by ammonium bicarbonate (ABC, 50 mM final concentration).
Samples were dried in a vacuum concentrator and resuspended in 6 M
urea/2 M thiourea. Disulfide bonds were reduced by dithiothreitol
(DTT, 2.5 mM, 50 °C for 15 min) and alkylated by iodoacetamid
(IAA, 5 mM, RT for 30 min in the dark). The samples were diluted to
2 M urea using 50 mM ABC and digested with trypsin (50:1 protein to
enzyme w/w ratio, 37 °C, overnight). Digestions were stopped
by adding 10% trifluoroacetic acid (TFA) until the pH was <2. Digests
were desalted using self-made C18 StageTips16 (link) and peptides were eluted using 80% acetonitrile and 20%, 0.1% TFA
in water. Eluates were dried and resuspended in 0.1% TFA. The sample
referred to as the pseudocomplex was obtained by mixing resuspended
peptides from all proteins in 1:1 molar ratio. The injected amount
was 1 μg of peptides.
using a 1:1 weight-to-weight (w/w) cross-linker to protein ratio.
C3b buffer was exchanged using 30 kDa molecular weight cutoff (MWCO)
filters (Millipore, Cork, Ireland) into cross-linking buffer (20 mM
HEPES, 20 mM NaCl, 5 mM MgCl2, pH 7.8). HSA, creatine kinase, myoglobin,
ovotransferrin, catalase, and cytochrome C were dissolved in cross-linking
buffer. Protein and cross-linker were mixed to a final concentration
of 1 mg/mL each. The mixture was incubated on ice for 2 h before the
reaction was stopped by ammonium bicarbonate (ABC, 50 mM final concentration).
Samples were dried in a vacuum concentrator and resuspended in 6 M
urea/2 M thiourea. Disulfide bonds were reduced by dithiothreitol
(DTT, 2.5 mM, 50 °C for 15 min) and alkylated by iodoacetamid
(IAA, 5 mM, RT for 30 min in the dark). The samples were diluted to
2 M urea using 50 mM ABC and digested with trypsin (50:1 protein to
enzyme w/w ratio, 37 °C, overnight). Digestions were stopped
by adding 10% trifluoroacetic acid (TFA) until the pH was <2. Digests
were desalted using self-made C18 StageTips16 (link) and peptides were eluted using 80% acetonitrile and 20%, 0.1% TFA
in water. Eluates were dried and resuspended in 0.1% TFA. The sample
referred to as the pseudocomplex was obtained by mixing resuspended
peptides from all proteins in 1:1 molar ratio. The injected amount
was 1 μg of peptides.
3
Purification and Analysis of Brain Proteins
4
Construction of Dox-Inducible Lentiviral Vectors
5
FASP-Based Tryptic Peptide Generation
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The tryptic peptides were generated using the filter-aided sample preparation (FASP) procedure as described previously (20 (link)) with minor modifications. In brief, 25 μL of the protein extracts were transferred onto 10 kDa molecular weight cut-off (MWCO) filters (Millipore, Bayswater, VIC, Australia) and washed twice using 100 μL of urea buffer (8 M urea in 0.1 M Tris-HCl, pH 8.2) with centrifugation (20,800 × g, 10 min). After which 100 μL of 25 mM iodoacetamide (in 8 M urea in 1 M Tris-HCl) was added for cysteine alkylation and the samples incubated in the dark at RT for 20 min. The excess iodoacetamide was then removed through two consecutive washes (100 μL of urea buffer) and centrifugation (20,800 × g, 10 min) steps. This was followed by buffer exchange with two 100 μL volumes of 100 mM ammonium bicarbonate (pH = 8.4) and centrifugation (20,800 × g, 10 min). Subsequently, 125 μL of 0.02 μg/μL sequencing grade trypsin (in 50 mM ammonium bicarbonate) was added to each filter and the samples were incubated overnight at 37°C. The digested peptides were collected in fresh centrifuge tubes with centrifugation at 20,800 × g for 15 min. The filters were washed with 200 μL 0.1% formic acid and the combined filtrates were evaporated to dryness in a vacuum centrifuge.
6
Isolation and Purification of Yeast Ribosomes
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Normal and SILAC-labeled yeast cells were collected by centrifugation at 4500 rpm for 10 min at 4°C, washed with cold deionized water and resuspended in 20 ml of lysis buffer (20 mM Tris-HCl pH 7.5, 140 mM NaCl, 5 mM MgCl2, 1 mM DTE) containing protease inhibitors (1 mM PMSF, 2 µg/ml aprotinin, 5 µg/ml pepstatin A, 1 µg/ml leupeptin). Cells were lysed with 5 to 6 cycles in French Press (Thermo Electron Corporation) at 18000 psi. Cell lysate was centrifuged twice at 15500 rpm for 15 min at 4°C in Type 50.2 Ti rotor (Beckmann). The supernatant was collected and the ribosomal particles were pelleted by centrifugation through a 20% sucrose cushion in buffer A (20 mM Tris-HCl pH 7.5, 250 mM NaCl, 5 mM MgCl2, 1 mM DTE) at 35000 rpm for 24 h (ω2t = 1.16*1012) at 4°C in in the same rotor. The pellet was resuspended in 2 ml of buffer A at 4°C for at least 2 hours, cleared at 15000 rpm for 20 min at 4°C in the same rotor and absorbance at 260 nm (A260) was measured. Thirty-five to forty A260 units (U) of ribosomal particles was carried onto a 10%–30% (w/w) sucrose gradient in buffer A and centrifuged at 20500 rpm for 16 h 15 min (ω2t = 2.7*1011) at 4°C in SW28 rotor (Beckmann). Fractions containing 80S ribosomal particles were collected, diluted 2 times with buffer A and concentrated using 100 000 Da MwCo filters (Millipore). Ribosomes were frozen in liquid nitrogen and stored at –80°C.
7
PEGylation and PEI Coating of MoS2 Nanosheets
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Ten milligrams of lipoic acid-modified PEG (LA-PEG) was added into 1 mg of MoS2 nanosheets dispersed in 2 mL of water. After sonication for 20 min and stirring overnight, excess PEG polymers were removed by centrifugal filtration with 100 kDa MWCO filters (Millipore, Billerica, MA, USA) and several times of water washing. The obtained MoS2-PEG or MoS2-PEG-FA were highly water-soluble and stored less than 4°C for use.
Generally, PEI used during this experiment was pre-dissolved in deionized water. One milligram of PEGylated MoS2 nanosheets and 0.1 mL PEI (50 mg/mL) were mixed in 2 mL deionized water. The mixture was stirred overnight under room temperature. Free PEI was removed by hyperfiltration.
Generally, PEI used during this experiment was pre-dissolved in deionized water. One milligram of PEGylated MoS2 nanosheets and 0.1 mL PEI (50 mg/mL) were mixed in 2 mL deionized water. The mixture was stirred overnight under room temperature. Free PEI was removed by hyperfiltration.
8
Glycoprotein Enrichment from Synovial Fluid
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Glycoprotein enrichment from 20 pooled synovial fluid samples containing 2.5 mg proteins was carried out by using a mixture of three agarose-bound lectins, Wheat Germ Agglutinin, Concanavalin A and Jacalin (Vector laboratories, USA), as described previously by our group [32 (link), 34 (link)]. Briefly, pooled lectin beads were incubated with protein samples in Tris-buffered saline (0.05 M Tris–HCl, pH 7.5, 0.15 M NaCl). After overnight incubation at 4 °C, the bound glycoproteins were eluted using competitive elution with a mixture of 100 mM of M-pyranoside, galactose, melibiose and N-acetyl glucoseamine in Tris-buffered saline, pH 7.5. The eluates were then washed and concentrated using 3 kDa MWCO filters (Amicon, Millipore, Ireland). The multilectin affinity approach yielded ~250 µg equivalent proteins and then stored at −20 °C until further use. These enriched proteins were then fractionated in SDS-PAGE followed by in-gel trypsin digestion.
9
Cross-linking of C3 and C3b Proteins
10
Quantitative Analysis of PDK1 Knockdown
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Lawns of HeLa cells were transfected with shRNAPDK1 or shRNAscrambled. Harvested cell pellets were lysed in lysis buffer (50 mM sodium phosphate pH 8, 300 mM NaCl, 0.5 mM DTT, 1% Nonidet P40) containing cocktails of protease inhibitors (Pierce Protease Inhibitor Mini Tablets #88666). Cell lysates were then concentrated (10 kDa MWCO Filters, Millipore #UFC501024) and run on 5–8% SDS-PAGE gels. Proteins were transferred to a PVDF membrane using a BioRad Semi Dry Transfer System. The PVDF membrane was blocked using LI-COR PBS Blocking Buffer (LI-COR #927–40100) and incubated overnight with primary antibodies (Rabbit anti-PDK1 (1:1000, Cell Signaling)), followed by a brief incubation with mouse anti-actin antibody (1:10000, Sigma). Subsequently, secondary antibodies conjugated with near-IR dyes were treated (Donkey anti-Rabbit-AlexaFluor790 (1:10,000, Jackson Immunoresearch), and Donkey anti-Mouse-DyLight690 (1:10,000, Cell Signaling)). The membranes were then imaged using a LI-COR Odyssey Sa Near-IR imager. Blots were analyzed using ImageStudioLite (LI-COR).
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