At first, the cell or tissue protein were extracted using the RIPA lysis buffer (P0013B, Beyotime). The protein concentration was determined by the BCA protein assay kit. All samples were prepared at a final concentration of 1 μg/μl in loading buffer (CW0027S, CwBio). Protein samples (20 μg) were loaded into a 10–15% SDS-polyacrylamide gel in the Bio-Rad Electrophoresis System to separate the proteins with different molecular weight. Then, the proteins in the gel were transferred to polyvinylidene difluoride (PVDF) membranes. After blocking in 5% bovine serum albumin (BSA) solution, the membranes were incubated with primary antibodies (Integrin-αL, ab186873, Abcam; Integrin-β2, ab131044, Abcam; C1q, ab71940, Abcam; Integrin-β3, ab75872, Abcam; CD11b, ab133357, Abcam; CD45, ab10558, Abcam; cleaved caspase-3, #9661s, Cell signaling technology; CD44, ab189524, Abcam; HO-1, ab68477, Abcam; β-tubulin, CW0098, CwBio; iNOS, ab15323, Abcam; CD206, ab125028, Abcam; Arginase-1, 93668s, Cell signaling technology; Histone H3, #9715, Cell signaling technology; GM130, 610,822, BD Biosciences) at 4 °C overnight. After incubation with the corresponding secondary antibodies, the PVDF membranes were imaged using Western chemiluminescent horseradish peroxidase (HRP) substrate (Millipore) with an imaging system (Tanon 4600, Shanghai).
Cw0027s
Manufactured by CWBIO
Sourced in China
The CW0027S is a laboratory equipment product. It is designed for the measurement and analysis of various samples. The core function of the CW0027S is to provide accurate and reliable data for research and testing purposes.
Automatically generated - may contain errors
Lab products found in correlation
Electrophoresis system, by Bio-Rad (2 mentions)
Ab133357, by Abcam (1 mentions)
Ab71940, by Abcam (1 mentions)
Ab75872, by Abcam (1 mentions)
Ab186873, by Abcam (1 mentions)
Ab131044, by Abcam (1 mentions)
Ab125028, by Abcam (1 mentions)
Ab189524, by Abcam (1 mentions)
Ab10558, by Abcam (1 mentions)
Ab68477, by Abcam (1 mentions)
Cw0098, by CWBIO (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Ab15323, by Abcam (1 mentions)
Chemidoc touch, by Bio-Rad (1 mentions)
Ab283655, by Abcam (1 mentions)
Ab86299, by Proteintech (1 mentions)
Ab283574, by Abcam (1 mentions)
Ab16502, by Abcam (1 mentions)
Western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Complete protease inhibitor cocktail, by Selleck Chemicals (1 mentions)
6 protocols using cw0027s
1
Comprehensive Protein Expression Analysis
2
Western Blot Analysis of Purified Viral Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The purified virus particles were mixed with reducing SDS-PAGE loading buffer (CWBIO, CW0027S) and heated in a boiling water bath for 5 min. Virion proteins were separated using a 10% SDS-polyacrylamide gel. Coomassie brilliant blue (Beyotime, P0017F) staining was performed for 30 min and then de-stained overnight. The protein bands were transferred to nitrocellulose (NC) membranes for western blot analysis. The membranes were blocked with 5% skim milk in PBS for 2 h at room temperature, and incubated overnight at 4°C with a 200-fold dilution of MR191 monoclonal antibody in blocking buffer. After washing with TBST, the NC membranes were incubated with an HRP-conjugated goat anti-human secondary antibody (Bioworld, BS10903) for 1 h at room temperature. After washing, electrochemiluminescence (ECL) substrate (Beyotime, P0018S) was added dropwise, and the bands were captured using a chemiluminescence imager (Bio-Rad, ChemiDoc Touch).
3
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein was then premixed with SDS-PAGE (CW0027S, CWBIO) in a 4:1 ratio and heated at 100 ℃ for 10 min. The protein samples were separated and transferred to polyvinylidene fluoride (PVDF) membrane. Then, the PVDF membrane was blocked with 5% nonfat milk for 1 h and incubated overnight with a primary antibody. Subsequently, the PVDF membranes were incubated with corresponding secondary antibodies at room temperature for 1 h and ECL Kit Chemiluminescence (S6008M, US EVERBRIGHT) was applied to visualize the immunoblots. Finally, ImageJ software was used for protein analysis.
The primary antibodies used in Western blot are as follows: anti-mas1 (1:1000, Proteintech, 20080–1-ap), anti-IL-1β (1:2000, Proteintech, 26,048–1-AP), anti-TNF-α (1:1000, Proteintech, 60291–1-Ig), anti iNOS (1:1000, Abcam, ab283655), anti-Arg-1 (1:1000, Abcam, ab283574), anti-p65(1:1000, Abcam, ab16502), anti-p65 (phospho S536) (1:1000, Abcam, ab86299), anti-TLR4(1:1000, Proteintech, 19811–1-AP), anti-IκB(1:2000, Proteintech, 10268–1-AP), anti-GAPDH (1:1000, Abcam, ab18602), anti-βactin (1:20000, Proteintech, 66009–1-Ig), anti-vinculin (1:10000, Proteintech, 66305–1-Ig).
The primary antibodies used in Western blot are as follows: anti-mas1 (1:1000, Proteintech, 20080–1-ap), anti-IL-1β (1:2000, Proteintech, 26,048–1-AP), anti-TNF-α (1:1000, Proteintech, 60291–1-Ig), anti iNOS (1:1000, Abcam, ab283655), anti-Arg-1 (1:1000, Abcam, ab283574), anti-p65(1:1000, Abcam, ab16502), anti-p65 (phospho S536) (1:1000, Abcam, ab86299), anti-TLR4(1:1000, Proteintech, 19811–1-AP), anti-IκB(1:2000, Proteintech, 10268–1-AP), anti-GAPDH (1:1000, Abcam, ab18602), anti-βactin (1:20000, Proteintech, 66009–1-Ig), anti-vinculin (1:10000, Proteintech, 66305–1-Ig).
4
Western Blot Analysis of cAB Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting analysis was performed to identify protein retention during the manipulation of cABs. All samples were prepared at a final protein concentration of 1 mg/ml in SDS-PAGE loading buffer (CW0027S, CwBio). Twenty microliters of sample was loaded into each well of a 10% SDS-polyacrylamide gel in a Bio-Rad Electrophoresis System. Protein staining was accomplished using Coomassie Blue Fast Staining solution, and the gels were destained in deionized water at 4°C overnight before imaging. For Western blot analysis, PVDF membranes were blocked with 5% BSA solution for 1 hour and then incubated with antibodies against CD3, CD8A, CD11b, CD44, and integrin-β3. Films were developed using western chemiluminescent HRP substrate (Millipore) with an imaging system (Tanon 5500, China).
5
Western Blot Analysis of Protein Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in lysis buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40 (Sigma, FlukaN74385) and 1% complete protease inhibitor cocktail (Selleck) on ice for 30 min. Cell lysates were mixed with 5x SDS loading buffer (CWBIO, CW0027S) at a ratio of 1:4 and boiled for 10 min at 95 °C. The boiled cell lysates were electrophoresed through 10% SDS-polyacrylamide gels and the separated proteins were transferred to PVDF membranes (Merck Millipore, Germany). After being blocked in 5% dry milk/TBST for an hour at RT, the membranes were incubated with diluted primary antibodies for 2 h at RT or overnight at 4 °C. Immunoreactive bands were detected using HRP-conjugated secondary antibody and ECL substrate (DiNing, DE2001) and visualized using FluorChem imager (ProteinSimple).
6
Quantifying STAT1 and STAT3 Activation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein expression of STAT1, p-STAT1, STAT3, and p-STAT3 were detected using a WB assay. Total protein was extracted from the MDSCs by using a radio-immunoprecipitation assay buffer (CW2334S; CWBIO, Jiangsu, China) and determined by a BCA protein assay (CW0014S; CWBIO, Jiangsu, China). 50 μg of protein was separated by 10% SDS-PAGE (CW0027S; CWBIO, Jiangsu, China) and then transferred into PVDF membranes. The membranes were blocked by 5% nonfat milk at room temperature for 1 hour, and then overnight at 4°C with the primary antibodies (SA00001-1; 1:2000; Proteintech Group, USA). The membranes were incubated at room temperature for 1 hour with secondary antibodies (SA00001-2; 1:2000; Proteintech Group, USA) the next day. The signal intensity was analyzed using the software Image J.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!