The recombinant RBD protein was mixed with 5 × loading buffer (Beyotime, Shanghai, China) and denatured for 5 mins at 100 °C. Denatured proteins (200 ng) were subjected to electrophoresis with 10% SDS-polyacrylamide gel and then transferred to PVDF membranes. After blocking by skim milk (Biofroxx), the membranes were incubated at 4 °C overnight, with the purified mAbs as primary Abs. The next days, the membranes were washed with TBST and incubated with HRP-conjugated mouse-anti-human Fc antibody (Abcam, ab99759, 1:10000) for 1 h at RT. The membranes were examined on ChemiDoc Imaging System (Bio-rad). The corresponding uncropped figures were included in the Supplementary Information .
Skim milk
Manufactured by neoFroxx
Sourced in Germany
Skim milk is a laboratory equipment used to separate milk into its components, specifically the removal of fat content. It utilizes a centrifugal force to separate the fat globules from the remaining milk components, resulting in a low-fat or non-fat milk product.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Loading buffer, by Beyotime (1 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (1 mentions)
Wbkls0500, by Merck Group (1 mentions)
Pierce bca protein assay reagent kit, by Thermo Fisher Scientific (1 mentions)
Bca protein assay kit, by Solarbio (1 mentions)
Bovine albumin, by Solarbio (1 mentions)
Enhanced chemiluminescence detection kit, by Keygen Biotech (1 mentions)
Ripa lysis buffer, by Keygen Biotech (1 mentions)
Bca protein quantitative kit, by Beyotime (1 mentions)
Nc membrane, by Merck Group (1 mentions)
Ripa lysate, by Beyotime (1 mentions)
Sodium dodecyl sulphate polyacrylamide gel electrophoresis, by Beyotime (1 mentions)
Np 40 lysis buffer, by Beyotime (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
5 protocols using skim milk
1
Recombinant RBD Protein Western Blot
2
Extraction and Western Blot Analysis of Liver Proteins
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Total proteins, nuclear and cytoplasmic proteins were extracted from liver tissues as described previously22 (link). In short, total protein and nuclear/cytoplasmic protein extracts of liver tissues were respectively homogenized with RIPA lysis buffer containing 1% 100 mmol/L PMSF (Cat# R0127, Biocolors, Shanghai, China) and NE-PER Nuclear and Cytoplasmic Extraction Reagents (Cat# 78835, Thermo Fisher Scientific). Protein concentration was determined using Pierce BCA Protein Assay Reagent Kit (Cat# 23225, Thermo Fisher Scientific). Proteins (30 μg/lane) were separated by 10% SDS-PAGE and transferred to NC membranes (Cat# 66485, Pall, New York, USA). The membranes were blocked using 5% skim milk (Cat# 1172GR500, BioFroxx, Einhausen, Germany) in TBST [Tris-buffered saline (TBS) containing 0.1% Tween-20] for 1 h at room temperature, and then incubated with different antibodies at 4 °C overnight. The blots were incubated by anti-rabbit IgG, HRP-linked antibody or anti-mouse IgG, HRP-linked antibody for 1 h at room temperature after washing with TBST three times the next day. Immunoreactive bands were visualized by chemiluminescence (Cat# WBKLS0500, Millipore, Burlington, VT, USA) following three washes with TBST.
3
Western Blot Analysis of Protein Expression
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Total protein was extracted from lung tissues or MRC-5 cells using RIPA lysis buffer (KeyGEN, China) containing proteinase and phosphatase inhibitor. The protein concentration was measured with the BCA protein assay kit (Solarbio, USA). Aliquots of 20-40 µg proteins were separated with 8%, 12%, or 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore, USA). After blocking with 5% skim milk (Biofroxx, Germany) or bovine albumin (Solarbio, USA), membranes were incubated with specific primary antibodies overnight at 4℃, then incubated with secondary antibodies at RT for 1.5 hours. Finally, an enhanced chemiluminescence detection kit (KeyGEN, China) was used to detect proteins and the ImageJ software was used for the grayscale analysis of protein bands. The antibodies used for western blotting are also listed in Table S1 .
4
Western Blot Protein Analysis Protocol
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Proteins were extracted from tissues or cells using RIPA lysate (Beyotime, Nanjing, China), and their contents were determined using the BCA protein quantitative kit (Beyotime, Nanjing, China). The protein samples were then subjected to sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (Beyotime, Nanjing, China) and transferred onto NC membranes (Millipore, Germany). The membranes were blocked with 5% skim milk (Biofroxx, Germany) at room temperature for 2 h, followed by incubation with the primary antibody overnight at 4°C. After washing, the membranes were incubated with a secondary antibody, Goat Anti‐Rabbit or Mouse (Li‐COR, USA). Finally, the protein bands were visualized and analysed by scanning the membranes using Odyssey (Li‐COR, USA) to determine their grey value.
5
Quantifying sFGL1 Protein Expression
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HEK 293T cells transfected with pcDNA3.1/V5-HisB-sFGL1 plasmid were collected at 48 h after transfection. After washing with PBS, cell samples were lysed with NP40 lysis buffer (Beyotime, China) and the concentration of the samples was determined using a BCA Protein Assay Kit. Equal amounts of protein were loaded and subjected to SDS-PAGE and then transferred to PVDF membranes (Millipore, USA) using a BIO-RAD Mini Trans-Blot Electrophoretic Transfer Cell. The membranes were blocked with 5% skim milk (BioFroxx, Germany) and then incubated with indicated primary antibodies overnight at 4°C, followed by HRP-linked secondary antibodies. Alpha-tubulin or GAPDH were used as a loading control. The protein bands were visualized using ChemiDoc™ MP Imaging System (Bio-Rad, USA). The experiment was repeated three times.
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