Cell extracts were prepared with M-PER (Pierce, Rockford, IL, USA) plus protease inhibitor cocktail (Halt™; Pierce, Rockford, IL, USA) and protein concentrations were determined using the BCA assay (Pierce, Rockford, IL, USA) [29 (link)]. Aliquots of protein lysates were separated on SDS-8 or 15% polyacrylamide gels and transferred to PVDF membrane filters, which were blocked with 5% blotting-grade milk (Bio-Rad, Hercules, CA, USA) in TBST (20 mM Tris-HCl (pH 7.6), 137 mM NaCl, 0.1% Tween 20) for 1 h. The filters were then incubated 1 h at room temperature with a 1:1000 dilution in TBST of antibodies against p16 (#92803, Cell Signaling Technology, Danvers, MA, USA), p21(sc-6246, Santa Cruz Biotechnology, Santa Cruz, CA, USA), IKK2 (GTX107970, GeneTex, Hsinchu, Taiwan) and NFκB p65 (phospho Ser536) (GTX133899, GeneTex, Taiwan), reacted with corresponding secondary antibodies, and detected using a chemiluminescence assay (Millipore, Billerica, MA, USA).
Blotting grade milk
Manufactured by Bio-Rad
Sourced in United States
Blotting grade milk is a laboratory reagent commonly used in Western blotting and other protein detection techniques. It is a protein-rich solution derived from dry milk powder that can be used to block nonspecific binding sites on membranes, reducing background signal and improving the specificity of target protein detection.
Automatically generated - may contain errors
Lab products found in correlation
Chemiluminescence assay, by Merck Group (5 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (2 mentions)
Tris buffer saline, by Boston BioProducts (2 mentions)
Tween 20, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by Santa Cruz Biotechnology (2 mentions)
Image lab, by Bio-Rad (2 mentions)
Chemidoc imager, by Bio-Rad (2 mentions)
Mammalian protein extraction agent, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Gtx133899, by GeneTex (1 mentions)
P21 sc 6246, by Santa Cruz Biotechnology (1 mentions)
Gtx107970, by GeneTex (1 mentions)
Anti p53, by Cell Signaling Technology (1 mentions)
Anti nanog, by Cell Signaling Technology (1 mentions)
M per, by Thermo Fisher Scientific (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Anti β catenin, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Anti c myc, by Cell Signaling Technology (1 mentions)
14 protocols using blotting grade milk
1
Western Blot Analysis of Cell Signaling
2
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
Cell extracts were prepared with mammalian protein extraction reagent (M‐PER; Thermo Fisher) plus protease inhibitor cocktail (Halt), and protein concentrations were determined by using the bicinchoninic acid assay (Thermo Fisher). Aliquots of protein lysates were separated on SDS‐10% polyacrylamide gels and transferred to polyvinylidene difluoride membrane filters, which were blocked with 5% blotting grade milk (Bio‐Rad, Hercules, CA, http://www.bio-rad.com ) in TBST (20 mM Tris‐HCl [pH 7.6], 137 mM NaCl, 1% Tween 20). Membranes were then probed with the indicated primary antibodies, reacted with corresponding secondary antibodies, and detected by using a chemiluminescence assay (EMD Millipore, Billerica, MA, http://www.emdmillipore.com ). Membranes were exposed to x‐ray film to visualize the bands (GE Healthcare Life Sciences, Piscataway, NJ, http://www.gelifesciences.com ). The primary antibodies included anti‐p53, anti‐Rb, anti‐Oct4, anti‐SOX‐2, anti‐Nanog, anti‐c‐Myc, anti‐β‐catenin, and anti‐β‐actin (1:1,000; Cell Signaling Technology, Beverly, MA, http://www.cellsignal.com ). The secondary antibodies included horseradish peroxidase‐conjugated donkey anti‐rabbit or anti‐mouse antibodies (1:2,000; GeneTex). The quantification of each protein expression level was normalized by internal control α‐tubulin, and the expression level of parental MSC was referred to as 1.
3
Western Blotting of HEK Cell Extracts
4
Western Blot Protein Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in 1% SDS extraction buffer supplemented with protease inhibitors (Santa Cruz Biotechnology, TX, USA). Briefly, cell extract was heated at 95°C for 5 mins, then sonicated (4 pulses, 4 cycles) to shear the DNA in the samples as previously described (1 (link)). Denatured samples (20–40 μg) were subjected to SDS-PAGE and proteins were transferred onto nitrocellulose membranes by electrophoretic transfer. Non-specific binding sites were blocked at room temperature for 1 h with 5% (w/v) Blotting-Grade milk (Bio-Rad Laboratories, CA, USA) in Tris–buffer saline (Boston Bio Products, MA, USA) containing 0.05% (v/v) Tween-20 (Thermo Fisher, MA, USA) (TBS-T). Membranes were incubated overnight with the primary antibodies, and then with the peroxidase-conjugated secondary antibody for 1 h (Supplementary Table 2 ). Signal was then captured by using Bio-Rad ChemiDoc imager, and band intensities were analyzed by densitometry on Image Lab (Bio-Rad Laboratories, CA, USA) or ImageJ software.
5
Evaluating EMT-associated Protein Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To evaluate the expression of EMT associated gene expression in protein levels, we used western blot in the study. Briefly, cells were lysed and protein was extracted using M-PER (Pierce, Rockford, IL, USA) plus protease inhibitor cocktail (Halt; Pierce). Ultrasonic the cell lysis in ice bath (300W for 4 seconds and rest for 8 seconds. Repeat three times.) was Protein concentrations were determined using BCA assay (Pierce). Aliquots of protein lysates were separated on SDS−polyacrylamide gels and transferred onto PVDF membrane, which was blocked with 5% blotting grade milk (Bio-Rad, USA) in PBST (0.1% Tween 20 in PBS). The membrane was then hybridized with the indicated primary antibodies to human E-cadherin, N-cadherin, β-catenin, vimentin, snail, slug and β-actin (Cell Signaling Technology, USA) followed by corresponding secondary antibodies conjugated with horseradish peroxidase, and then detected using a chemiluminescence assay (Millipore, USA). Membranes were exposed to X-ray film to visualize the bands. The band of β-actin was used as endogenous control.
6
Quantifying EGFR and STAT3 activation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed and proteins were extracted using mammalian protein extraction agent (ThermoFisher Scientific Pierce, Waltham, MA, USA) plus Halt protease inhibitor cocktail (ThermoFisher Scientific Pierce). Protein concentrations were determined using bicinchoninic acid (BCA) protein assay kit (ThermoFisher Scientific Pierce). Aliquots of protein lysates were separated on sodium dodecyl sulfate−polyacrylamide gels and transferred onto a nitrocellulose membrane, which was blocked with 5% blotting grade milk (Bio-Rad, Hercules, CA, USA) in PBST (0.1% Tween 20 in phosphate-buffered saline [PBS]). The membrane was then hybridized with the indicated primary antibodies specific to human EGFR, phospho-EGFR (Tyr1068), STAT3, and phospho-STAT3 (Tyr705), then with the corresponding secondary antibodies conjugated with horseradish peroxidase, and detected using a chemiluminescence assay (EMD Millipore, Temecula, CA, USA). Membranes were exposed to X-ray film (Kodak China Investment, Shanghai, People’s Republic of China) to visualize the bands. β-Actin was used as loading control. All primary and secondary antibodies used in the Western blotting were purchased from Cell Signaling Technology (Danvers, MA, USA).
7
Protein Expression Analysis of H9c2 Cardiomyocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The H9c2 or primary cardiomyocytes were grown in 6-well plates, divided into the four groups as described above, and treated for 24 h. Subsequently, the cells were collected for protein extraction and lysed in RIPA buffer (1% NP-40, 1 mmol/l Na3VO4, 1 mmol/l NaF, and 0.5 mmol/l PMSF) on ice for 30 min. The lysate was clarified by centrifugation, and the supernatant was removed. The protein concentrations were assessed by using the BCA Protein Assay Kit (Pierce), and the absorbance was read at 490 nm by means of ELISA reader. Cell lysate containing 30 μg of total protein was separated by 10% SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride membranes. The membranes were blocked through incubation in 5% blotting grade milk (Bio-Rad) in TBS-T (0.1% Tween 20 in TBS) and then probed overnight with the following primary antibodies: anti-STAT-phospho Y705 (Abcam), Bcl-2 (CST), Bax (CST), and β-actin at 4°C. Subsequently, the membrane was incubated with HRP-conjugated secondary antibodies (CST). The fluorescence signals were visualized by using SuperSignal West Pico Chemiluminescent Substrate (Pierce).
8
Western Blot Protein Extraction and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell extracts were prepared using the M-PER protein extraction reagent (Pierce, Rockford, IL) plus protease inhibitor cocktail (HaltTM; Pierce) and protein concentrations were determined using the bicinchoninic acid (BCA) assay (Pierce). Aliquots of protein 20–40 μg lysates were separated on SDS–10% polyacrylamide gels and transferred to PVDF membrane filters. Membranes were blocked with 5% blotting grade milk (Bio-Rad, Hercules, CA) in TBST (20 mM Tris–HCl [pH 7.6], 137 mM NaCl, 1% Tween 20). Membranes were then probed with the indicated primary antibodies, reacted with the corresponding secondary antibodies, and results detected using a chemiluminescence assay (Millipore, Billerica, MA). Membranes were exposed to X-ray film to visualize the bands (Amersham Pharmacia Biotech).
9
Immunoblotting Analysis of EGFR and STAT3
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed and protein was extracted using mammalian protein extraction agent (ThermoFisher Scientific Pierce, Waltham, MA, USA) plus Halt protease inhibitor cocktail (ThermoFisher Scientific Pierce). Protein concentrations were determined using a bicinchoninic acid assay (ThermoFisher Scientific Pierce). Aliquots of protein lysates were separated on sodium dodecyl sulfate‐polyacrylamide gels (SDS‐PAGE) and transferred onto a nitrocellulose membrane, which was blocked with 5% blotting grade milk (BioRad, Hercules, CA, USA) in PBST (0.1% Tween 20 in PBS). The membrane was then hybridized with primary antibodies to human EGFR and phospho‐EGFR (Tyr1068). We also tested the level of STAT3 and phospho‐STAT3 (Tyr705) as STAT3 has been identified to regulate PD‐L1 transcriptionally. Then, with the corresponding secondary antibodies conjugated with horseradish peroxidase, and detected using a chemiluminescence assay (EMD Millipore, Temecula, CA, USA), membranes were exposed to X‐ray film (Kodak China Investment, Shanghai, China) to visualize the bands. β‐actin was used as loading control. All primary and secondary antibodies used in the Western blotting were purchased from Cell Signaling Technology (Danvers, MA, USA).
10
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After drug-treating time came to 24 h, cells were collected for protein extraction. Cells were lysed in RIPA buffer (1% NP-40, 1 mmol/l Na3VO4, 1 mmol/l NaF, 0.5 mmol/l PMSF) on ice for 30 min. Lysate was abandoned by centrifugation while the supernatant was removed. Protein concentrations were assessed using the BCA Protein Assay Kit (Pierce) and the absorbance was read at 490 nm by means of ELISA reader. Cell lysate containing 30 μg of total protein was run on 10% SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride membranes. The membrane was blocked with 5% blotting grade milk (Bio-Rad) in TBS-T (0.1% Tween 20 in TBS) and then probed with the following primary antibodies: Nanog (Abcam), PD-L1 (Abcam), OCT-4 (CST), SOX-2 (CST), and β-actin (CST) at 4°C. Next day, the membrane was incubated with HRP-conjugated secondary antibodies (CST). Fluorescence signal was visualized using SuperSignal West Pico Chemiluminescent Substrate (Pierce).
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!