C3b proteolytic degradation regulatory activity of mossMFHR1 and plasmaFH was compared in a fluid phase cofactor assay modified from Michelfelder et al. (2017) (link). Briefly, in a 20 μl reaction, increasing doses of mossMFHR1 or plasmaFH, and corresponding maximal volume of the parental plant Δxt/ft purified extract as control, were incubated with 2 μg C3b and 500 ng complement factor I (FI) (CompTech, Texas, USA) in PBS at 37°C for 30 min. The reaction was stopped by the addition of sample loading buffer (Bio-Rad) under reducing conditions (50 mM DTT, NuPAGE™, Thermo Fisher Scientific). The FH and FI catalyzed proteolytic cleavage of C3b into iC3b was analyzed by visualizing the α-chain cleavage fragments α’68 and α’43 by SDS-Page in 10% SDS–polyacrylamide gels and Coomassie staining. The remaining intact α-chain was quantified by band densitometry (Quantity One, Bio-Rad).
Sample loading buffer
Manufactured by Bio-Rad
Sourced in United States
Sample loading buffer is a liquid solution used to prepare samples for gel electrophoresis. It contains dyes and other components that facilitate the loading of samples into the gel and the visualization of the separated molecules.
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Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Chemidoc mp imaging system, by Bio-Rad (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Tris 2 carboxyethyl phosphine hydrochloride, by Bio-Rad (2 mentions)
Reducing agent, by Bio-Rad (2 mentions)
Nupage, by Thermo Fisher Scientific (1 mentions)
Quantity one, by Bio-Rad (1 mentions)
Foxo3a, by Cell Signaling Technology (1 mentions)
Protein a g, by Thermo Fisher Scientific (1 mentions)
T8787, by Merck Group (1 mentions)
Anti flag m2 affinity gel, by Merck Group (1 mentions)
Pierce anti c myc agarose, by Thermo Fisher Scientific (1 mentions)
Non fat dry milk, by Bio-Rad (1 mentions)
Hrp conjugated goat anti mouse or goat anti rabbit secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Ab83690, by Abcam (1 mentions)
β actin antibody, by Merck Group (1 mentions)
Ibright cl1500, by Thermo Fisher Scientific (1 mentions)
Las 4000, by Fujifilm (1 mentions)
19 protocols using sample loading buffer
1
Comparative Analysis of Complement Regulation
2
Muscle Tissue Protein Expression Analysis
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Snap-frozen GAs muscle tissues were homogenized in lysis buffer and prepared in sample loading buffer (Bio-Rad, Hercules, CA, USA). Lysate proteins were separated on a 10% SDS-PAGE gels and then transferred to polyvinylidenedifluoride (PVDF) membranes (Merck Millipore, Danvers, MA, USA). After blocking in TBS buffer containing 5% non-fat dry milk for 1 h at room temperature, the membranes were incubated and gently shaken overnight at 4 °C with primary antibodies. After washing with TBS, the membranes were incubated with secondary antibodies for 1 h at room temperature with shaking. After washing, the protein bands were detected and analyzed by a ChemiDoc™ MP Imaging System (Bio-Rad, Hercules, CA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the loading control. The results are expressed as the integrated optical density relative to GAPDH. Primary antibodies against GAPDH were used as the loading control. The results are expressed as the integrated optical density relative to GAPDH. Primary antibodies against p-ULK1(ser555), ULK1, p-AMPK (Thr172), AMPK, LC3B, p-p38 MAPK(Thr180/Tyr182), p38 MAPK, and FoxO3a were purchased from Cell Signaling Technology (Danvers, MA, USA). Primary antibody against GAPDH was from Proteintech Group, Inc. (Chicago, IL, USA).
3
Western Blot Analysis of Cell Signaling
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Samples were prepared in sample loading buffer (Bio-Rad). The lysates were separated on SDS-PAGE gels and transferred to PVDF membranes (Merck Millipore, Danvers, MA, USA). After blocking, the membranes were incubated overnight with the following primary antibodies: p-E-cadherin (S838 + S840), E-cadherin, p-Erk1/2 (Thr202/Tyr204), Erk1/2, p-p38 MAPK (Thr180/Tyr182), p38 MAPK, p-S6RP (Ser235/236), S6RP, catalase, SOD2, GPX1, VDAC, TOM20, COX IV, Tfam, p-AMPK (Thr172), and β-actin. Then the membranes were incubated with secondary antibodies and detected using the ChemiDoc MP Imaging System (Bio-Rad).
4
Immunoprecipitation and Western Blot Analysis
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After the designed treatments, the cells were suspended with the immunoprecipitation (IP) lysis buffer (10 mM Tris-HCl, pH 7.4 [Vivantis Technologies, PB0852], 100 mM NaCl [Vivantis Technologies, PB0570], 2.5 mM MgCl2, 0.5% Triton X-100 [Sigma-Aldrich, T8787], phosphatase inhibitor and proteinase inhibitor cocktail), followed by sonication and centrifuged at 12,000 g for 10 min. Part of the supernatant (1–1.5 mg) was transferred to a new tube and diluted with the IP lysis buffer to achieve the concentration at 1–1.5 μg/μl. The supernatant was incubated with either control IgG or antibodies following the instructions of antibody datasheets overnight with gentle rotation at 4°C and was subsequently incubated with 10 μl protein A/G (Thermo Fisher Scientific, 20,421) for additional 4 h. For FLAG IP or MYC IP, the supernatant was incubated with 10 μl anti-FLAG M2 Affinity Gel (Sigma-Aldrich, A2220) or Pierce™ Anti-c-Myc Agarose (Thermo Fisher Scientific, 20,168) and mixed overnight with gentle rotation at 4°C. Next, the immunoprecipitates were washed with IP lysis buffer 3 times. The immunoprecipitants were then eluted by boiling for 5 min in sample loading buffer (Bio-Rad Laboratories, 1,610,737) and analyzed with immunoblotting.
5
Mitochondrial and Metabolic Protein Analysis
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Liver tissues were homogenized and prepared in sample loading buffer (Bio-Rad Laboratories, Inc.). Total protein was separated using 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (EMD Millipore). After blocking with 5% non-fat dry milk (Bio-Rad Laboratories, Inc.), the membranes were incubated at 4˚C overnight with the following primary antibodies: G6Pase (Abcam, ab83690, 1:1,000), transcription factor A mitochondrial (TFAM; EMD Millipore, ABE483, 1:1,000), cytochrome c oxidase IV (COX IV), translocase of the outer mitochondrial membrane 20 (TOM20), voltage-dependent anion channel (VDAC), pyruvate dehydrogenase (PDH), phosphoenolpyruvate carboxykinase (PCK2), branched-chain α-keto acid dehydrogenase complex (BCKDH) and β-actin. COX IV (cat. no. #4844, 1:1,000), TOM20 (#13929, 1:1,000), VDAC (#4661, 1:1,000), PDH (#2784, 1:1,000), PCK2 (#6924, 1:1,000) and BCKDH (#90198, 1:1,000) antibodies were purchased from Cell Signaling Technology, Inc. and β-actin antibody was from MilliporeSigma (A5441, 1:2,000). The membranes were then incubated with HRP-conjugated Goat anti-Rabbit or Goat anti-Mouse secondary antibodies (Invitrogen; Thermo Fisher Scientific, Inc.; Cat. nos. 65-6120 or 62-6520, respectively; all 1:2,000) and detected by the ChemiDoc™ MP Imaging System (Bio-Rad Laboratories, Inc.).
6
Protein Expression Analysis in Brain Tissues
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To measure the expression levels of proteins, the whole brain and sectioned hippocampus and cerebral cortex tissues were homogenized by mixing with ProteinEx animal cell/tissue buffer (Gene All Biotechnology, Seoul, Republic of Korea) containing 1% protease inhibitor cocktail (Quartett, Berlin, Germany) and centrifuged at 13,000× g for 10 min at 4 °C. Afterwards, the supernatant was mixed with sample loading buffer (#1610374, Bio-Rad) and used for immunoblotting analysis. Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and proteins separated according to size were transferred to a PVDF membrane (Millipore, Billerica, MA, USA). After that, it was treated with 5% skim milk for 1 h to block the membrane and then washed three times for 10 min each using tris-buffered saline (TBS) with 0.1% of Tween 20 (TBST buffer). The membrane and primary antibody (1:1000) were incubated at 4 °C for 12 h. Then, the secondary antibody (1:2500) was reacted with the membrane for 1 h at room temperature, washed three times for 10 min with TBTS buffer, and the luminescent band was chemically analyzed using an image analyzer (iBright CL1500, Invitrogen, Waltham, MA, USA). Also, antibody information used in this study is shown in Table 1 below.
7
Western Blot Protein Analysis Protocol
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The cells were collected and lysed in sample loading buffer (Bio-Rad, Hercules, CA, USA). Total protein concentrations were quantified using a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Quantified proteins were loaded onto SDS-polyacrylamide gels and separated by molecular weight via electrophoresis (PAGE). Then, proteins were transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA); 5% BSA in Tris-buffered saline containing 0.1% Tween-20 (TBST) was used for blocking. The membranes were blocked for 30 min at room temperature with mild shaking, then incubated with antibodies diluted in 2.5% BSA in TBST overnight at 4 °C with mild shaking. Following secondary antibody incubation and washing with TBST, the chemiluminescence signals were detected using the WEST-Queen detection system (iNtRON Biotechnology, Seongnam, Korea) and LAS-4000 (Fuji Film Corp., Tokyo, Japan).
8
1D SDS-PAGE Venom Protein Analysis
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The 1D gel method utilised the following specific conditions: 1 mm 12% SDS-Page gels with resolving gel layer (3.3 mL Milli-Q H2O, 4 mL 30% acrylamide mix (Bio-Rad, Hercules, CA, USA), 2.5 mL 1.5MTris-HClbuffer (Tris—Sigma-Aldrich, St. Louis, MO, USA; HCl—Univar, Wilnecote, UK), pH 8.8, 100 μL 10% SDS (Sigma-Aldrich, St. Louis, MO, USA), 4 μL TEMED (Bio-Rad, Hercules, CA, USA), 100 μL 10% APS (Bio-Rad, Hercules, CA, USA); 20 μg venom sample per lane after dissolving in 3 μL of 4× sample loading buffer (Bio-Rad, Hercules, CA, USA) brought up to 12 μL total volume, with DTT (Sigma-Aldrich, St. Louis, MO, USA); reducing conditions were 3 min incubation at 100 °C; gels were run at room temperature for at 120 V (Mini Protean3 power-pack from Bio-Rad, Hercules, CA, USA) for 20 min and then 140 V for 60 min; runs were stopped when dye front was less than 10 mm from the base of the gel. Gels were stained with colloidal coomassie brilliant blue G250 (34% methanol (VWR Chemicals, Tingalpa, QLD, Australia), 3% orthophosphoric acid (Merck, Darmstadt, Germany) 170 g/L ammonium sulfate (Bio-Rad, Hercules, CA, USA), 1 g/L coomassie blue G250 (Bio-Rad, Hercules, CA, USA)) overnight and then destained in 1% acetic acid (Univar, Wilnecote, UK).
9
Western Blot Analysis of RIP3 and MLKL
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For Western blot analysis of RIP3, cells were lysed in 1.25× sample buffer [83 mM tris (pH 6.8), 6.7% SDS, 13.3% glycerol, 1.3% β-mercaptoethanol, and 0.03% bromophenol blue] and boiled at 100°C for 5 min before being subjected to SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis, as previously described (46 (link)). PVDF (polyvinylidene difluoride) membranes were blocked with 5% skim milk, followed by incubation with RIP3 (ProSci Inc., 1:500) or HSP90 (Santa Cruz Biotechnology, 1:1000) antibodies. For analysis of MLKL protein, cells were lysed in ice-cold M2 lysis buffer [50 mM NaF, 20 mM tris (pH 7.0), 0.5% NP-40, 250 mM NaCl, 3 mM EDTA, and 3 mM EGTA, supplemented with Roche protease and phosphatase inhibitor cocktails] for 30 min before centrifugation to pellet insoluble component as described (47 (link)). Samples were mixed 3:1 with 4× sample loading buffer (Bio-Rad) before SDS-PAGE and Western blot analysis. PVDF membranes were blocked in 5% bovine serum albumin and probed with MLKL (Millipore, 1:500) or GAPDH (glyceraldehyde phosphate dehydrogenase) (Millipore, 1:1000) antibodies. Goat anti-mouse (1:2500), anti-rabbit (1:5000), or anti-rat (1:2500) IRDye secondary antibodies (Rockland) were used. The protein bands were visualized using Odyssey Infrared Imaging System (LI-COR Biotechnology).
10
Western Blot Analysis of Protein Expression
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The compound-treated cells were lysed in a 2× sample loading buffer (Bio-Rad, Hercules, CA, USA). Protein concentrations were quantified by the bicinchoninic acid assay (BCA) method [60 (link)]. On SDS-PAGE gel, proteins were separated and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). After blocking with 5% bovine serum albumin (BSA) in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h shaking, the membrane was probed with antibodies for anti-NDRG1 (#9485; 1:1000), anti-cyclin D1 (#55506; 1:1000), anti-CDK4 (#12790; 1:1000), anti-CDK6 (#13331; 1:1000), anti-Bcl-2 (#15071; 1:1000), anti-E-cadherin (#14472; 1:1000), anti-vimentin (#5741; 1:1000), anti-Snail (#3879; 1:1000), anti-MMP9 (#13667; 1:1000) (Cell Signaling Technology, Beverly, MA, USA), anti-beta-actin (#47778; 1:10,000), anti-Bax (#7480; 1:500) (Santa Cruz Biotechnology, Dallas, TX, USA), and anti-N-cadherin (#610920; 1:1000) (BD Biosciences, San Jose, CA, USA). Specific proteins were detected by secondary antibodies, and the chemiluminescence signals were detected using a Miracle-StarTM Western Blot Detection System (iNtRON Biotechnology, Seongnam, Republic of Korea). Image J (National Institutes of Health, Bethesda, MA, USA) was used for the quantitative analysis.
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