For western blotting of total lysates, cells were harvested and washed three times in 1× PBS and lysed in RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS, 500 µM dithiothreitol) with protease inhibitors (Sigma-Aldrich). Approximately 20 μg of whole cell lysates were loaded in Novex WedgeWell 4-20% Tris-Glycine Gels (Invitrogen) in a Tris-Glycine-SDS buffer (Invitrogen) and separated by gel electrophoresis (SDS-PAGE). The proteins were then transferred to Immun-Blot PVDF membranes (Thermo Fisher Scientific) for antibody probing. Membranes were incubated with 10% bovine serum albumin in TBS with 3% Tween 20 (TBST) for 30 min at room temperature (RT), then incubated for variable times with suitable antibodies diluted in 5% bovine serum albumin in 1× TBST, washed with TBST and incubated with a dilution of 1:10,000 of the secondary antibody for 1 h at RT. The antibody was visualized using Super Signal West Dura Extended Duration Substrate (Thermo Fisher Scientific) and imaged with Amersham Imager 680.
Immunblot pvdf membranes
Manufactured by Thermo Fisher Scientific
ImmunBlot PVDF membranes are a type of laboratory equipment used for protein transfer and detection in Western blotting applications. These membranes are made of polyvinylidene fluoride (PVDF) material and provide a stable and reliable platform for the immobilization of proteins, allowing for their subsequent analysis and identification.
Automatically generated - may contain errors
Lab products found in correlation
Tris glycine sds buffer, by Thermo Fisher Scientific (3 mentions)
Novex wedgewell 4 20 tris glycine gel, by Thermo Fisher Scientific (3 mentions)
Amersham imager, by Cytiva (3 mentions)
Super signal west dura extended duration substrat, by Thermo Fisher Scientific (2 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (1 mentions)
Amersham imager 680, by Cytiva (1 mentions)
3 protocols using immunblot pvdf membranes
1
Western Blotting of Whole Cell Lysates
2
Western Blot Analysis of Cell Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For total lysate, cells were harvested and washed three times in 1× PBS and lysed in radioimmunoprecipitation assay buffer (RIPA buffer) (50 mM Tris-HCl pH7.5, 150 mM NaCl, 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS, 500 μM DTT) with proteases inhibitors. Twenty μg of whole cell lysate were loaded in Novex WedgeWell 4–20% Tris-Glycine Gel (Invitrogen) and separated through gel electrophoresis (SDS–PAGE) Tris-Glycine-SDS buffer (Invitrogen). The proteins were then transferred to ImmunBlot PVDF membranes (ThermoFisher) for antibody probing. Membranes were incubated with 10% BSA in TBST for 30 min at room temperature (RT), then incubated for variable times with the suitable antibodies diluted in 5% BSA in 1× TBST, washed with TBST, and incubated with a dilution of 1:10000 of secondary antibody for one hour at RT. The antibody was then visualized using Super Signal West Dura Extended Duration Substrat (ThermoFisher) and imaged with Amersham Imager 680. Full membranes (uncropped) of the blots presented in the main figures are available as source data file.
3
Western Blot Protein Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For total lysate, cells were harvested and washed three times in 1X PBS and lysed in RIPA buffer (50mM
Tris-HCl pH7.5, 150mM NaCl, 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS, 500uM DTT) with proteases inhibitors. Twenty μg of whole cell lysate were loaded in Novex WedgeWell 4-20% Tris-Glycine Gel (Invitrogen) and separated through gel electrophoresis (SDS-PAGE) Tris-Glycine-SDS buffer (Invitrogen).
The proteins were then transferred to ImmunBlot PVDF membranes (ThermoFisher) for antibody probing.
Membranes were incubated with 10% BSA in TBST for 30 minutes at room temperature (RT), then incubated for variable times with the suitable antibodies diluted in 5% BSA in 1X TBST, washed with TBST and incubated with a dilution of 1:10000 of secondary antibody for one hour at RT. The antibody was then visualized using Super Signal West Dura Extended Duration Substrat (ThermoFisher) and imaged with Amersham Imager 680. Used antibodies are listed in the "Antibodies" section of the methods.
Tris-HCl pH7.5, 150mM NaCl, 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS, 500uM DTT) with proteases inhibitors. Twenty μg of whole cell lysate were loaded in Novex WedgeWell 4-20% Tris-Glycine Gel (Invitrogen) and separated through gel electrophoresis (SDS-PAGE) Tris-Glycine-SDS buffer (Invitrogen).
The proteins were then transferred to ImmunBlot PVDF membranes (ThermoFisher) for antibody probing.
Membranes were incubated with 10% BSA in TBST for 30 minutes at room temperature (RT), then incubated for variable times with the suitable antibodies diluted in 5% BSA in 1X TBST, washed with TBST and incubated with a dilution of 1:10000 of secondary antibody for one hour at RT. The antibody was then visualized using Super Signal West Dura Extended Duration Substrat (ThermoFisher) and imaged with Amersham Imager 680. Used antibodies are listed in the "Antibodies" section of the methods.
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!