For Western immunoblotting, equal amounts (15 µg/lane) of proteins were diluted in RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA, United States) and mixed with Laemmli buffer (120 mM Tris-HCl, 20% glycerol, 0.4% SDS, and 0.02% bromophenol blue, pH 6.8) containing fresh 5% β-mercaptoethanol (Sigma Aldrich, Saint Louis, MO, United States). The samples were denatured at 99°C for 7 min. The proteins were separated on 10% SDS-PAGE gels and then blotted onto polyvinylidene difluoride (PVDF; BioRad Laboratories, Hercules, CA, United States) membranes. The membranes were blocked with either 5% non-fat dried milk (Santa Cruz Biotechnology, Dallas, TX, United States) or BSA (Sigma Aldrich, Saint Louis, MO, United States) in TBS-T (20 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH 7.6) for 1 h at room temperature with agitation. The membranes were incubated with primary antibodies (listed below) overnight at 4°C, followed by incubation with alkaline phosphatase-linked goat antirabbit or antimouse antibodies for 1 h at RT. The membranes were washed three times in TBS-T for 5 min. The bands were visualized using 1-Step™ NBT/BCIP Substrate Solution (Thermo Fisher Scientific, Waltham, MA, United States) and their intensities were semiquantitatively measured with ImageJ software (https://imagej.nih.gov/ij/ ). All experiments were run in triplicates.
Polyvinylidene difluoride pvdf
Manufactured by Bio-Rad
Sourced in United States
Polyvinylidene difluoride (PVDF) is a type of synthetic polymer membrane material commonly used in laboratory equipment. It serves as a support medium for various analytical and separation techniques, particularly in the field of Western blotting, where it is used to immobilize and transfer proteins for further detection and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Thermo Fisher Scientific (4 mentions)
Non fat dried milk, by Santa Cruz Biotechnology (2 mentions)
1 step nbt bcip substrate solution, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
β mercaptoethanol, by Merck Group (2 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Gel logic 2200 pro, by Carestream (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Roche (1 mentions)
Chemiluminescence imager, by LI COR (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Cell culture reagents, by Euroclone (1 mentions)
Fitc annexin 5 apoptosis detection kit, by BioLegend (1 mentions)
Chemiluminescent substrate and secondary antibody 32460, by Thermo Fisher Scientific (1 mentions)
Xevo g2 xs qtof, by Waters Corporation (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Silica gel 60 f254, by Merck Group (1 mentions)
Bradford reagent, by Bio-Rad (1 mentions)
Non fat dry milk, by Merck Group (1 mentions)
P fak, by Cell Signaling Technology (1 mentions)
11 protocols using polyvinylidene difluoride pvdf
1
Western Blotting Protocol for Protein Analysis
2
Western Blotting for Protein Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For Western immunoblotting, equal amounts (5–20 µg/lane) of protein were diluted in RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) and mixed with Laemmli buffer (120 mM Tris-HCl, 20% glycerol, 0.4% SDS, and 0.02% bromophenol blue, pH 6.8) containing fresh 5% β-mercaptoethanol (Sigma Aldrich, Saint Louis, MO, USA). The samples were denatured at 95 °C for 7 min. The proteins were separated on 7.5–10% SDS-PAGE gels and then blotted onto polyvinylidene difluoride (PVDF; BioRad Laboratories, Hercules, CA, USA) membranes. The membranes were blocked with either 5% non-fat dried milk (Santa Cruz Biotechnology, Dallas, TX, USA) or BSA (Sigma Aldrich, Saint Louis, MO, USA) in TBS-T (20 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH 7.6) for 1 h at room temperature with agitation. The membranes were incubated with primary antibodies overnight at 4 °C, followed by incubation with alkaline phosphatase-linked goat antirabbit or antimouse antibodies for 1 h at RT. The membranes were washed three times in TBS-T for 5 min. The bands were visualized using 1-Step™ NBT/BCIP Substrate Solution (Thermo Fisher Scientific, Waltham, MA, USA) and their intensities were semiquantitatively measured with ImageJ software (https://imagej.nih.gov/ij/ ). All experiments were run in triplicates.
3
Quantifying Protein Expression in PC12 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For protein expression analyses, PC12 cells cultured in 60 mm petri-dishes (Sarstedt, Nürnbrecht, Germany) were lysed in 100 μl of RIPA buffer supplemented with 1 mM PMSF (Roche Diagnostics, Mannheim, Germany). Protein concentrations were determined using the BCA protein assay (Pierce, Rockford, IL, USA). Protein lysates (20 μg) were heated for 5 min at 94 °C in Laemmli sample buffer containing 5% β-mercaptoethanol and then loaded on 4–15% Tris-glycine SDS-PAGE gels, then transferred electrophoretically onto polyvinylidene difluoride (PVDF; Bio-Rad, Hercules, CA, USA) membranes at 200 mAmp for 2 h. Membranes were blocked with 5% non-fat dry milk for 1 h and incubated overnight at 4 °C with the antibodies specific to Shootin1, ERK1/2, p-ERK1/2 (Thr202/Tyr204), Akt, p-Akt (Thr308) and GAPDH (CST, Danvers, MA, USA). Protein bands were detected with horseradish peroxidase-conjugated anti-rabbit secondary antibodies (CST; Danvers, MA, USA) and visualized by West-Femto ECL reagents (Pierce; Rockford, IL, USA). Chemiluminescent signals of immunoblots were documented using Gel Logic 2200 Pro (Carestream Health; Rochester, NY, USA). The net intensity of specific proteins was quantified using ImageJ (NIH, Bethesda, MD, USA).
4
Western Blot Protein Expression Analysis
5
Synthesis and Apoptosis Evaluation of Novel Compounds
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The materials and reagents used in the synthetic procedures were purchased from Sigma Aldrich Co. (Stenheim, Germany) and used without purification. All solvents were analytically pure and dried before use. TLC was carried out on aluminum sheets precoated with silica gel 60 F254 (Merck). Column chromatography was performed using silica gel 60 (230–400 mesh).
High-resolution MS (HRMS) ESI analyses were performed on a Xevo G2-XSQTof (Waters) mass spectrometer. Mass spectrometric detection was performed in the in the positive ion mode. The 1H and 13C NMR spectra were recorded at 400 and 100 MHz, respectively, on an Agilent Technologies 400 MHz Premium Shielded spectrometer. Chemical shifts (δ) are reported in ppm relative to TMS and coupling constants (J) in Hz. Cell culture reagents were obtained from Euroclone (Milan, Italy). Chemical reagents and propidium iodide (PI) were obtained from Sigma Aldrich (St. Louis, MO, USA). The Annexin V-FITC apoptosis detection kit was obtained from Biolegend (San Diego, CA, USA). Chemiluminescent substrate and secondary antibody (32460) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Bradford reagent and polyvinylidene difluoride (PVDF) membranes were obtained from Bio-Rad (Hercules, CA, USA).
High-resolution MS (HRMS) ESI analyses were performed on a Xevo G2-XSQTof (Waters) mass spectrometer. Mass spectrometric detection was performed in the in the positive ion mode. The 1H and 13C NMR spectra were recorded at 400 and 100 MHz, respectively, on an Agilent Technologies 400 MHz Premium Shielded spectrometer. Chemical shifts (δ) are reported in ppm relative to TMS and coupling constants (J) in Hz. Cell culture reagents were obtained from Euroclone (Milan, Italy). Chemical reagents and propidium iodide (PI) were obtained from Sigma Aldrich (St. Louis, MO, USA). The Annexin V-FITC apoptosis detection kit was obtained from Biolegend (San Diego, CA, USA). Chemiluminescent substrate and secondary antibody (32460) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Bradford reagent and polyvinylidene difluoride (PVDF) membranes were obtained from Bio-Rad (Hercules, CA, USA).
6
Western Blot Analysis of EMT Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After ovalitenone at non-toxic concentrations (0-200 µM) treatment, cells were incubated on ice for 40 min with RIPA buffer, 1% Triton X-100, 100 mM PMSF, and a protease inhibitor. Cell lysates were analyzed for protein content using the BCA protein assay kit from Pierce Biotechnology (Rockford, IL). Equal amounts of protein samples (approximately 60–100 µg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) (Bio-Rad Laboratories Inc., CA, USA) or nitrocellulose membranes (Bio-Rad Laboratories Inc., CA, USA). The resulting blots were blocked for 2 h with 5% (w/v) non-fat dry milk (Merck, DA, Germany) in TBS-T (Tris-buffer saline with 0.1% Tween containing 25 mM Tris-HCl (pH 7.5), 125 mM NaCl, and 0.1% Tween 20) and incubated with the specific primary antibodies against N-cadherin, E-cadherin, Snail, Slug, p-mTOR, mTOR, p-FAK, FAK, p-AKT, AKT, Cdc42, and β-actin (Cell Signaling, Danvers, MA, USA) at 4 °C overnight. After three washed with TBS-T, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling, Danvers, MA, USA) for 2 h at room temperature. Finally, the immune blots were detected by enhanced chemiluminescence (Supersignal West Pico; Pierce, Rockford, IL, USA) and quantified using ImageJ software (NIH, Bethesda, MD, USA).
7
Rabbit Polyclonal Antibodies against S. oneidensis ScyA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rabbit polyclonal antibodies against S. oneidensis ScyA were prepared in accordance with standard protocols by Genscript (Nanjing, China) and used for the immunoblotting analyses. Pellets of mid-log phase cells were washed once with phosphate-buffered saline (PBS), and resuspended in 2x Laemmli SDS sample buffer. After boiled for 5 min, samples were loaded onto 10% SDS-PAGE and either stained with Coomassie Brilliant Blue or electrophoretically transferred to polyvinylidene difluoride (PVDF) according to the manufacturer's instructions (Bio-Rad). Processing of the immunoblots was performed essentially as described previously (Dong et al. 2012 (link)). For estimation of relative abundance of proteins in the gel, band intensity was measured using ImageJ software (NIH).
8
Evaluating OBA-RT Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A549 cells were seeded at a density of 4 × 105 cells/well in 6-well plates overnight. Cells were treated with OBA-RT (0–25 µM) for 24 h. Then, cells were washed with cold 1X PBS and incubated in RIPA buffer, 1% Triton X-100, 100 mM PMSF, and a protease inhibitor for 30 min on ice. Protein concentrations were quantified using BCA protein assay kit from Pierce Biotechnology (Rockford, IL, USA). Cell lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) (Bio-Rad Laboratories Inc., Hercules, CA, USA). The membrane was blocked with 5% (w/v) non-fat dry milk power (Merck, Darmstadt, Germany) at room temperature for 2 h and each membrane was incubated with the specific primary antibodies for overnight at 4 °C, as well as incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling, Danvers, MA, USA) for 2 h at room temperature. The protein expression was observed using chemiluminescence (Supersignal West Pico; Pierce, Rockford, IL, USA) and quantified using ImageJ software (NIH, Bethesda, MD, USA).
9
TCDD and ITE Signaling Pathway
10
Comprehensive Antibody Panel for Oligodendrocyte Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary antibodies for β-actin (#12620), phospho-mitogenactivated protein kinase 1 and 2 (MEK1/2) (Ser217/221) (#9154), MEK1/2 (#9126), phospho-extracellular signal-regulated kinase 1 and 2 (ERK1/2) (Thr202/Tyr204) (#4370), ERK1/2 (#9102), myelin-associated glycoprotein (MAG) (#9043), myelin basic protein (MBP), #78896), and gliomaassociated oncogene homolog 1 (GLI1) (#2643) were from Cell Signaling Technology. Antibodies for 2',3'-cyclic-nucleotide 3'phosphodiesterase (CNPase) (SC-166558) and myelin oligodendrocyte glycoprotein (MOG) (SC-376138) were from Santa Cruz Biotechnology. Antibodies for oligodendrocytes transcription 2 (Olig2) (AB35128) and neuron-glial antigen 2 (NG2) (AB15894) were from Millipore. Antibodies for proteolipid protein (PLP) (ab28486) and platelet-derived growth factor receptor α (PDGFRα) (ab203491) were from Abcam Inc. The antibody for (sex determining region Y)-box 10 (Sox10) (MBS3201449) was from MyBioSource. Secondary antibodies, IHC Detection Reagents (HRP, Rabbit #8114), 3,3'-diaminobenzidine tetrahydrochloride (DAB) Substrate Kit (#8059), and hematoxylin (#14166) were obtained from Cell Signaling Technology. Polyvinylidene difluoride (PVDF), membranes, and molecular weight standards for Western blot were obtained from BIO-RAD. The enhanced chemiluminescence Western blotting system was from Thermo Fisher Scientific Inc.
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!