Triplicate cultures of SU-1, JW12, and JW13 were grown in YES liquid medium for 40 h under standard conditions, harvested, and disrupted with a mortar and pestle under liquid nitrogen, and a protein extract was prepared as previously described [2 (link)]. Total protein concentrations were measured using the Pierce® BCA Protein Assay Reagent (Thermo Scientific, Waltham, MA, USA). 100 μg of total proteins per lane was separated by SDS-PAGE using a 4–20% gradient Tris-HCl Ready Gel® (Bio-Rad, Hercules, CA, USA) and transferred to polyvinylidene difluoride (PVDF) membranes (PerkinElmer Life Sciences, Waltham, MA, USA). Membranes were exposed to polyclonal anti-AtfB antibodies (YSR, 5 μg/mL) and incubated with goat anti-rabbit secondary antibodies conjugated to the fluorescent tag IRDye 800 CW (Li-Cor Biosciences, Lincoln, NE, USA) as previously described [2 (link)]. For aflatoxin enzymes, highly specific antibodies against Nor-1, OmtA, and Ver-1 were used as previously described [46 (link),49 ,50 (link),51 (link)]. Visualization of proteins was performed using an Odyssey infrared imaging system (Li-Cor Biosciences) at 795 nm, as previously described [2 (link)].
Polyvinylidene difluoride pvdf membranes
Manufactured by PerkinElmer
Sourced in United States
Polyvinylidene difluoride (PVDF) membranes are a type of laboratory equipment used for various applications. These membranes are made of a hydrophobic polymer material, PVDF, which provides high chemical and mechanical resistance. PVDF membranes are commonly used for filtration, separation, and transfer processes in various research and analytical settings.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using polyvinylidene difluoride pvdf membranes
1
Quantitative Western Blot Analysis of Aflatoxin Biosynthesis Enzymes
2
Analyzing Metalloproteinase Enzymes MMP-2 and MMP-9
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All chemicals including quercetin were purchased from Sigma (St. Louis, MO)
unless specified otherwise. Antibodies for MMP-2 and MMP-9 were purchased from
Abcam (Burlingame, CA). Polyvinylidenedifluoride (PVDF) membranes and enhanced
chemiluminescence (ECL) detection reagents were bought from Perkin Elmer Life
Sciences, Inc. (Waltham, MA).
unless specified otherwise. Antibodies for MMP-2 and MMP-9 were purchased from
Abcam (Burlingame, CA). Polyvinylidenedifluoride (PVDF) membranes and enhanced
chemiluminescence (ECL) detection reagents were bought from Perkin Elmer Life
Sciences, Inc. (Waltham, MA).
3
SDS-PAGE and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells grown in six-well plates and treated as per experimental requirement were washed with PBS and lysed with 300 ml of SDS sample buffer (3% 2-mercaptoethanol, 20% glycerine, 0.05% bromophenol blue, 3% SDS). Cell lysates were collected and boiled at 95 C with 1000 rpm shaking for 7 minutes. Samples were stored at À20 C until required. SDS-PAGE and western blotting were performed as described earlier (Gurumurthy et al., 2010) . Briefly, proteins from the cell lysates were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membranes (PerkinElmer Life Sciences), and blocked with 3% milk powder in Tris-buffered saline (containing 0.5% Tween 20) for 30 min before incubation with the appropriate antibodies.
The bound primary antibodies were incubated with the corresponding HRP-conjugated secondary antibodies. Immunoreactive proteins were detected on an X-ray film directly after addition of ECL reagent (Amersham Biosciences).
The bound primary antibodies were incubated with the corresponding HRP-conjugated secondary antibodies. Immunoreactive proteins were detected on an X-ray film directly after addition of ECL reagent (Amersham Biosciences).
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!