The RIPA buffer (Invitrogen, Waltham, MA, USA) that was supplemented with protease and phosphatase inhibitors (Roche, South San Francisco, CA, USA) was used to break GC cells. After quantification with a BCA protein assay kit (Pierce, Waltham, MA, USA), samples of protein (20 μg) were separated by SDS-PAGE gel, and transferred to nitrocellulose membrane (Invitrogen, Waltham, MA, USA), which was blocked in 5% non-fat milk (Bio-Rad, Hercules, CA, USA) at room temperature for 1 h, reacted with the primary antibodies in 5% non-fat milk at 4 °C overnight, and incubated with secondary antibodies (in 5% non-fat milk) at room temperature for 1 h. The blots were developed using Super Signal Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA) and were visualized with Las4000 system (GE Healthcare, Chicago, IL, USA). The densitometric quantification was performed using ImageJ software.
Non fat milk
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
Non-fat milk is a laboratory reagent used in various scientific experiments. It serves as a source of protein, carbohydrates, and other nutrients for cell cultures, microbiological assays, and other applications where a standardized nutrient source is required.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (3 mentions)
Anti rabbit igg 7074, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (2 mentions)
Coating buffer, by ImmunoChemistry Technologies (2 mentions)
96 well plate, by Thermo Fisher Scientific (2 mentions)
Human iga, by Merck Group (2 mentions)
Inactivated goat serum, by Thermo Fisher Scientific (2 mentions)
Mouse anti human iga hrp, by Abcam (2 mentions)
Bm blue substrate, by Roche (2 mentions)
Spectramax m2 elisa plate reader, by Molecular Devices (2 mentions)
Goat anti human igg hrp, by Southern Biotech (2 mentions)
Tween 20, by Merck Group (2 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Hrp conjugated mouse anti rabbit igg, by Bioworld Technology (1 mentions)
Hrp conjugated anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Psd 95, by Thermo Fisher Scientific (1 mentions)
Iblot2, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
11 protocols using non fat milk
1
Western Blot Analysis of GC Cells
2
SUMO Protein Expression Dynamics
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At 0, 4, 8, 12, 24, and 48 h post-infection, protein lysate samples were boiled for 10 min in sodium dodecyl sulfate (SDS) buffer. The samples were then separated on a 12% SDS-polyacrylamide gel electrophoresis gel and electro-transferred to a nitrocellulose membrane (Millipore, USA) for 40 min. The membrane was blocked with 5% non-fat milk (Thermo Scientific, USA) for 1 h, and then incubated with rabbit anti-SUMO1 or Ubc9 monoclonal antibodies (Abcam, UK) diluted in Tris-buffered saline with Tween 20 (TBST) buffer for 1 h at 37°C. The membrane was washed with TBST buffer 3 times and incubated with HRP-conjugated mouse anti-rabbit IgG (Bioworld, USA) overnight at 4°C. After 3 washes, the membrane was stained using an HRP-DAB kit (Zhongshan Golden Bridge, China).
3
Western Blot for Synaptic Proteins
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SVs (10–20 µg) from each animal were loaded into 4–12% Bis-Tris wells (Invitrogen, Waltham, MA, USA) under reducing conditions, followed by transfer to a nitrocellulose membrane using iBlot2 (Invitrogen) and immunodetection. Nonfat milk (5%) was used to block nonspecific antibody binding (Thermo Fisher Scientific, Waltham, MA, USA). After blocking, membranes were incubated overnight at 4 °C with a primary antibody. Primary antibodies included GAPDH (Invitrogen), PSD95 (Invitrogen), GFAP (Sigma-Aldrich, St. Louis, MO, USA), SYP (ThermoFisher), VGLUT1 (Santa Cruz Biotech, Dallas, TX, USA), and SNAP25 (Synaptic Systems, Goettingen, Germany). MEGF8 (Bioworld Technology, St. Louis Park, MN, USA) and LAMTOR4 (Cell Signaling, Danvers, MA, USA) were additionally selected from the proteomic analysis for post-validation. Secondary antibodies were HRP-conjugated anti-rabbit IgG (Thermo Scientific) and HRP-conjugated anti-mouse IgG (Thermo Scientific). Primary and secondary antibody dilutions were done according to the manufacturer’s suggestion and are shown in Supplementary Table S1 . Blots were developed using Azure cSeries Imager (Azure Biosystems, Dublin, CA, USA) with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific).
4
Western Blot Analysis of Liver Proteins
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To extract the proteins, liver tissue cells and Hep3B cells were lysed in radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Inc.) supplemented with Mini protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA). The protein concentration was determined using bicinchoninic acid kit (Thermo Fisher Scientific, Inc.). Whole cell lysate (50 µg) was separated on 10% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. Membranes were blocked for 1 h at 25°C using 4% non-fat milk (Thermo Fisher Scientific, Inc.) and probed with anti-GAPDH antibody (cat. no. ab9458; 1:1,000); anti-phosphorylated (p)-p65 antibody (cat. no. ab76302; 1:500); anti-total p65 antibody (cat. no. ab207297; 1:500); anti-IκBα antibody (cat. no. ab55341; 1:500; all Abcam, Cambridge, UK) at 4°C for 12 h. Membranes were then incubated with goat anti-rat IgG horseradish peroxidase-conjugated antibody (cat. no. ab205720; 1:2,000; Abcam) for 1 h at 25°C. Blots were visualized using enhanced chemiluminescence reagent (Thermo Fisher Scientific, Inc.). GAPDH was used to confirm equal loading.
5
Western Blot Analysis of Protein Regulation
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Proteins were extracted using radioimmunoprecipitation assay (RIPA) buffer (Thermo, Waltham, MA). Equal amounts of the total proteins were separated with sodium dodecyl sulfate-PAGE (SDS–PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Thermo). After blocking with 5% nonfat milk (Thermo) for 2 h, the membranes were incubated with primary antibodies at 4 °C overnight and then with secondary antibodies at room temperature for 2 h. The membranes were washed with 0.1% Tris-buffered saline with Tween (TBST) and subjected to chemiluminescence analysis using the enhanced chemiluminescence (ECL) kit (CWBIO, Beijing, China). The antibodies against myocilin (sc-515500) and GAPDH (sc-25778) were purchased from Santa Cruz Biotechnology (Dallas, TX). Antibodies against Grp78 (3177), Grp94 (2104), PDI (2446), calnexin (2679), XBP1s (27,901), CHOP (2895), eIF2α (5324), phosphorylated eIF2α (3398), cleaved caspase 3(9664), cleaved caspase 9 (7237), and IRE1 (3194) were purchased from Cell Signaling Technology (Boston, MA). Phosphorylated IRE1 antibody was purchased from Abcam (ab124945, Cambridge, MA). All secondary antibodies, anti-mouse immunoglobulin (IgG; 7076) and anti-rabbit IgG (7074), were purchased from Cell Signaling Technology.
6
Western Blot Analysis of Ocular Proteins
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Radioimmunoprecipitation buffer (Thermo, Waltham, United States) was used for protein extraction. 40 μg total proteins, 10 μL supernatant or 4 µl aqueous humor were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Thermo). The membrane was then blocked in nonfat milk (Thermo) for 2 h. After incubation with primary antibodies at 4°C overnight and with secondary antibodies at room temperature for 2 h, the membranes were washed with 0.1% tris-buffered saline with Tween and subjected to chemiluminescence analysis using the enhanced chemiluminescent reagent kit (CWBIO, Beijing, China). The antibodies against myocilin (sc-515500, sc-137233) and GAPDH (sc-25778) were purchased from Santa Cruz biotechnology (Dallas, TX, United States). Antibodies against Grp78 (3177), Grp94 (2104), p62 (5114), LC3B (43566), mTOR (2983) and p-mTOR (2974) were purchased from Cell Signaling Technology (Boston, MA, United States). All the secondary antibodies, anti-Mouse IgG (7076) and anti-Rabbit IgG (7074), were purchased from Cell Signaling Technology.
7
Ad5 Neutralization Assay for Plasma Antibodies
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An Ad5 neutralization assay was carried out using participant plasma obtained one week previous to sigmoidoscopy as previously described38 (link).
For ELISA, 96 well plates (Thermo Scientific) were coated overnight with 1×109 viral particles of Ad5 purified by double-banding, CsCl gradients, and dialysis, and diluted in 100μl of coating buffer (Immunochemistry Technologies). Plates were then blocked for 2h with 5% non-fat milk (Fisher), 3% Inactivated Goat Serum (Invitrogen), and 0.2% Tween-20 (Sigma) in 1× PBS (Life Tech). Explant samples were assayed undiluted and at 1:10 and 1:100 dilutions in blocking buffer, and negative controls included RPMI with 20% FBS. Plasma samples were diluted in six 1:5 dilutions starting with 1:500. Samples, standard curves, and controls were incubated for 2h at 37°C.
After washing, goat anti-human IgG-HRP (Southern Biotech) or mouse anti-human IgA-HRP (Abcam) at 1:1,000 was incubated for 1h at 37°C, then detected with addition of BM Blue substrate (Roche) for 15 min. The reaction was stopped by adding 50μl of 1N H2SO4 and read within 20min using a SpectraMax M2 ELISA Plate Reader (Molecular Devices) at 450nm absorbance. Standard curves used human IgG (Bethyl) or human IgA (Sigma), at 20.0 to 0.009ng/well, and the linear ranges were used for extrapolation of the concentrations.
For ELISA, 96 well plates (Thermo Scientific) were coated overnight with 1×109 viral particles of Ad5 purified by double-banding, CsCl gradients, and dialysis, and diluted in 100μl of coating buffer (Immunochemistry Technologies). Plates were then blocked for 2h with 5% non-fat milk (Fisher), 3% Inactivated Goat Serum (Invitrogen), and 0.2% Tween-20 (Sigma) in 1× PBS (Life Tech). Explant samples were assayed undiluted and at 1:10 and 1:100 dilutions in blocking buffer, and negative controls included RPMI with 20% FBS. Plasma samples were diluted in six 1:5 dilutions starting with 1:500. Samples, standard curves, and controls were incubated for 2h at 37°C.
After washing, goat anti-human IgG-HRP (Southern Biotech) or mouse anti-human IgA-HRP (Abcam) at 1:1,000 was incubated for 1h at 37°C, then detected with addition of BM Blue substrate (Roche) for 15 min. The reaction was stopped by adding 50μl of 1N H2SO4 and read within 20min using a SpectraMax M2 ELISA Plate Reader (Molecular Devices) at 450nm absorbance. Standard curves used human IgG (Bethyl) or human IgA (Sigma), at 20.0 to 0.009ng/well, and the linear ranges were used for extrapolation of the concentrations.
8
Western Blot Analysis of Whole Cell Lysates
9
Ad5 Neutralization Assay for Plasma Antibodies
10
Extracellular Vesicle Protein Analysis
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RIPA lysis buffer (Beyotime) was applied for extracting the total protein. After measurement of protein concentration, protein (about 40 µg/lane) was subjected to SDS-PAGE (Beyotime). Subsequently, the proteins were transferred to nitrocellulose membranes (Invitrogen), which were then blocked with 5% non-fat milk (Yili, Beijing, China) and incubated with primary antibody overnight at 4°C. The primary antibodies including cluster of differentiation 63 (CD63; 1:1000, ab118307), cluster of differentiation 9 (CD9; 1:500, ab223052), TPD52 (1:5000, ab155296), and GAPDH (1:2000, ab37168) were bought from Abcam (Cambridge, UK). After that, the corresponding secondary antibody (1:4000, ab205718, Abcam) was used for the combination with the primary antibody. At last, the visualization of protein blots was achieved by an enhanced chemiluminescence reagent (Solarbio, Beijing, China).
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