Cellular protein samples were isolated with a Protein Extraction Kit (Qiagen, GmbH, Hilden, Germany) according to the kit's manual and supplemented with a protease inhibitor cocktail (Abcam, Cambridge, UK). Protein samples were separated with 10% SDS-PAGE gel and were transferred to a polyvinylidene fluoride hydrophobic membrane (Millipore, Bedford, MA, USA). The membrane was blocked with 5% skimmed milk (Solarbio, Beijing, China) overnight at 4°C to cover the nonspecific binding sites. Then the membrane was inoculated with the rabbit anti-mouse KLF-4 (BM0485, Abzoom Biolabs, Dallas, TX, USA; 1 : 300), PAI-1 (sc-8979, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1 : 200), E-cadherin (sc-7870, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1 : 500), Collagen I (ab21286, Abcam, Cambridge, UK; 1 : 200), mouse anti-mouse α-Smooth Muscle Actin (α-SMA) (sc-53142, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1 : 200), or rabbit anti-mouse β-actin (sc-7210, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1 : 800) at room temperature for 2 hours. The specific binding to each protein marker was presented with the incubation with the peroxidase-conjugated secondary antibody (Promega, Madison, WI, USA) and the electrochemiluminescence (ECL) detection system (Amersham, Uppsala, Sweden). The protein level was presented as a ratio to β-actin.
Skimmed milk
Manufactured by Solarbio
Sourced in China
5% skimmed milk is a laboratory reagent used for various applications in scientific research and analysis. It serves as a standard solution for calibrating and testing equipment, as well as a component in media preparation for cell culture studies. The product provides a consistent, low-fat milk solution for these purposes.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Beyotime (3 mentions)
Ripa lysis buffer, by Solarbio (3 mentions)
Eyoecl plus, by Beyotime (2 mentions)
Pvdf membrane, by Solarbio (2 mentions)
Loading buffer, by Solarbio (2 mentions)
Peroxidase conjugated secondary antibody, by Promega (1 mentions)
Protease inhibitor cocktail, by Abcam (1 mentions)
Ecl detection system, by Cytiva (1 mentions)
Protein extraction kit, by Qiagen (1 mentions)
Polyvinylidene fluoridehydrophobic membrane, by Merck Group (1 mentions)
Sc 8979, by Santa Cruz Biotechnology (1 mentions)
Sc 7210, by Santa Cruz Biotechnology (1 mentions)
Sc 53142, by Santa Cruz Biotechnology (1 mentions)
E cadherin, by Santa Cruz Biotechnology (1 mentions)
Bca protein assay kit, by Solarbio (1 mentions)
E cadherin, by Thermo Fisher Scientific (1 mentions)
N cadherin, by Thermo Fisher Scientific (1 mentions)
Xcell 2 blot module, by Thermo Fisher Scientific (1 mentions)
Surepage gels, by Beyotime (1 mentions)
β actin, by Affinity Biosciences (1 mentions)
11 protocols using skimmed milk
1
Protein Expression Analysis Protocol
2
Western Blot Analysis of EMT Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
As described by Chen et al and his colleagues [25 (link)], after 48 h of transfection, cells were harvested for protein extraction. Proteins were quantified with a BCA protein assay kit (Solarbio). Then, an aliquot of 20 μg protein was electrophoresed on SurePAGE gels (Beyotime) and transferred onto nitrocellulose membranes using XCell II Blot Module (Thermo Fisher). The membranes were subjected to incubation with the antibodies for N-cadherin (1:500; Thermo Fisher), E-cadherin (1:1000; Thermo Fisher), SPARCL1 (1:1000; Thermo Fisher), and β-actin (1:3000; Affinity, Nanjing, China) after being blocked with 5% skimmed milk (Solarbio). The nitrocellulose membranes were incubated with secondary antibodies (1:3000; Affinity). At last, a Bio-Rad image analysis system was performed to develop protein blots with eyoECL Plus (Beyotime). The protein expression was normalized to β-actin.
3
Western Blot Analysis of Metalloproteinases
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The tissue and cell samples were lysed using NP-40 buffer (Beyotime), and proteins were degenerated by heating. Then, an aliquot of 20 μg protein samples were electrophoresed on 10% bis–tris-acrylamide gel (Thermo Fisher), followed by the wet-transfer with the nitrocellulose membranes (Pall Life Science, Beijing, China). After blocking in skimmed milk (Solarbio), the membranes were probed with anti-matrix metalloprotein 9 (anti-MMP9; 1:1000; Affinity, Nanjing, China), anti-MMP2 (1:1000; Affinity), anti-MAP3K3 (1:1000; Affinity) and anti-GAPDH (1:8000; Affinity). After that, the secondary antibody against goat anti-rabbit IgG (1:5000; Affinity) was incubated with the membranes. The protein signals were detected with eyoECL Plus (Beyotime). GAPDH was employed to normalize protein abundance.
4
Western Blot Protein Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted from HGFs using ice-cold radioimmunoprecipitation (RIPA) lysis buffer (Solarbio). After being quantified by BCA (Thermo Fisher Scientific), the protein samples were mixed with loading buffer (Solarbio), separated by electrophoresis on SDS-PAGE. The proteins in the gel were transferred on a polyvinylidene fluoride (PVDF) membrane (Beyotime). The membranes were blocked with 5% skimmed milk (Solarbio) and incubated overnight at 4°C with primary antibody. The membranes were washed with Tris-buffered saline and incubated with secondary antibody for 90 min at room temperature. The PVDF membranes were subjected to chemiluminescence detection using an ECL Western Blotting Detection Kit (Solarbio).
5
Detecting TLR-4 Expression by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cytosolic proteins were isolated using a standard cell lysis buffer (Cell Signaling Technology Inc., Danvers, MA, USA), and supplemented with a protease inhibitor cocktail (Roche Biochemicals, Basel, Switzerland). Protein samples were separated on a gel (12%) using sodium dodecyl sulfate polyacrylamide gel electrophoresis; these were transferred to a polyvinylidene difluoride hydrophobic membrane (Millipore, Bedford, MA, USA). Nonspecific binding sites on the membrane were blocked overnight with 5% skimmed milk (Solarbio, Beijing, China) at 4°C; the membrane was then inoculated with a rabbit polyclonal antibody against TLR-4 (Abcam, Cambridge, UK) or GAPDH (Sinobio, Beijing, China) at room temperature for 2 h; subsequently, the membrane was stained with a peroxidaseconjugated secondary antibody against rabbit IgG (Pierce, Rockford, IL, USA) at room temperature for 1 h. Finally, specific binding was visualized by chemiluminescence by using the enhanced chemiluminescence western blotting detection reagent (Amersham, Uppsala, Sweden). TLR-4 expression (gray color) was determined relative to that of GAPDH.
6
Western Blot Analysis of Protein Targets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed using a radio immunoprecipitation assay (RIPA) buffer (Boster, Wuhan, China) containing a protease inhibitor. The total protein was quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher, Waltham, MA, USA) following the procedure outlined in the instructions. Proteins were separated using 12.5% polyacrylamide gel and transferred to 0.45 μm of polyvinylidene fluoride (PVDF) membranes using a Trans-blot Turbo device (Bio-Rad, Hercules, CA, USA) at 75 V on ice for 1.5 h. The membranes were blocked with 5% skimmed milk (Solarbio, Beijing, China) for 1 h at room temperature. Then, the primary antibody was used to incubate the membranes overnight at 4 °C. The primary antibody and dilution rate are described as follows: anti-NRAMP1 (1:500) (Novus, Wuhan, China), β-actin (1:20,000) (Proteintech, 66009-1-Ig, Rosemont, IL), caspase-3 (1:1000) (Abclonal, Wuhan, China), proliferating cell nuclear antigene (PCNA) (1:5000) (Elabscience Biotechnology Co. Ltd., Wuhan, China). Next, the membranes were incubated with the secondary antibody horse radish peroxidase (HRP) goat anti-rabbit IgG (1:10,000) and HRP goat anti-mouse IgG (1:10,000) (ABclonal, Wuhan, China) at room temperature for 1 h. Finally, the membranes were scanned using chemiluminescence imaging software (AI600 Control. RDP).
7
Quantifying Protein Expression via Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot was applied to analyze protein expression. In brief, denatured protein samples (30 µg) were separated by 12% SDS-PAGE, transferred to PVDF membranes (EMD Millipore), blocked in 5% skimmed milk (Beijing Solarbio Science & Technology, Co., Ltd.) for 2 h at room temperature, washed with PBS containing Tween-20 (PBST) and incubated with primary antibodies [total OXPHOS human WB antibody cocktail (cat no. ab110411); anti-myc tag (cat no. ab206486); anti-Flag (cat no. ab205606); and anti-6×His tag (cat no. ab18184); all 1:1,000 dilution; Abcam] at 4°C overnight on an orbital shaker. PVDF membranes were then washed with PBST and incubated with horseradish peroxidase-conjugated anti-mouse IgG (cat no. sc-2005; 1:1,000 dilution; Santa Cruz Biotechnology, Inc.) at room temperature for 1 h prior to washing with PBST and adding a working solution of Enhanced Chemiluminescence Reagent Kit (Beyotime Institute of Biotechnology). Membranes were imaged using the iBright FL1000 Imaging System (Invitrogen; Thermo Fisher Scientific, Inc.). Image J software 1.53a (National Institutes of Health) was used to quantify the gray value of western blot bands and the protein expression level was normalized as the gray value of the target protein/the loading control protein.
8
Western Blot Analysis of Signaling Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The 143B cells were inoculated in cell culture dishes and divided into 4 groups and treated with free Res, Res/Lps, and FA-Res/Lps. Cells were extracted from protein by RIPA lysis buffer containing a mixture of protease and phosphatase inhibitors and protein concentrations were measured using the BCA method. Use SDS-PAGE gels for protein separation. Protein samples were transferred to PVDF membranes, closed with 5% skimmed milk (Solarbio, Beijing), washed twice with TBST and then treated with appropriately diluted primary antibodies (p-JAK2, diluted 1:1000; JAK2, diluted 1:1000; p-STAT3, diluted 1:1000; STAT3, diluted 1:1000; BCL-2. diluted 1:1000; Bax, diluted 1:1000; Caspase3, diluted 1:1000; Cleaved Caspase3, diluted 1:1000; Cytochrome, diluted 1:1000; N-Cadherin, diluted 1:1000; Vimentin, diluted 1:1000; Tubulin, diluted 1:1000) overnight at 4°C. After washing three times with TBST, samples were incubated with secondary antibodies for 1 h at room temperature and developed using ECL illumination solution (Millipore, USA).
9
Western Blot Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in lysis buffer (BC3711, Solarbio), and then the concentration was determined by BCA. An equal volume of protein (40 µg) was separated on 8% SDS-PAGE and transferred onto PVDF membranes (Thermo Fisher Scientific, Rockford, IL, USA). After blocked by 5% skimmed milk (Solarbio), the membranes were cultured with primary antibodies at 4°C for 16 h, and then with secondary antibody for at 20°C for 2 h. The immunoblots were developed by Western ECL substrate (PIERCE), and the gray value was measured by a Gel-Pro-Analyzer Software.
10
Western Blot Analysis of Stem Cell Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed with RIPA lysis buffer (Solarbio) on ice for 15 mins and centrifugated at 10000 rpm/min at 4°C for 30 mins. The concentration of protein from cell supernatant was quantified using the BCA regent. The equal amount of protein samples was loaded on SDS-PAGE and then transferred to PVDF membrane (Beyotime), blocked with 5% skimmed milk (Solarbio) at room temperature for 2 h and incubated with primary antibody at 4 °C overnight. Primary antibodies were listed as: anti-METTL14 (HPA038002, SigmaAldrich), anti-NANOG (4893, CST), anti-β-catenin (sc-7963, Santa Curz). Afterwards, the PVDF membrane was washed for 3 times with PBS, incubated with primary antibody at room temperature for 1.5 h and the chemiluminescence was detected by Pierce™ ECL Western Blotting Substrate (32106, Thermo Fisher Scientific).
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!