Undigested protein extracts from each of the in vitro translation reactions were boiled in 1× LDS buffer (Invitrogen), resolved on a 4–12% Bis-Tris denaturing/reducing SDS-PAGE (Invitrogen) and transferred onto a nitrocellulose membrane (Bio-Rad) for immunoblotting. Membranes were then blocked with 5% non-fat dry milk (Safeway) in PBS-tween buffer and probed with a primary antibody targeting SJ GST (GE 27–4577–01) as an epitope. Incubations with the primary antibody were done at 4 °C overnight using a 1:1000 dilution. Secondary incubations were performed in 5% non-fat dry milk in PBS-tween using 1:10000 diluted peroxidase-conjugated rabbit anti-goat IgG (H+L) (Pierce). Membranes were visualized using an ECL plus western blotting kit (Amersham) and detected with radiographic film (Thermo).
Ecl plus western blotting kit
Manufactured by Cytiva
The ECL plus western blotting kit is a laboratory equipment product used for the detection and analysis of proteins in western blot experiments. It provides a chemiluminescent detection system to visualize and quantify the proteins of interest on a membrane.
Automatically generated - may contain errors
2 protocols using ecl plus western blotting kit
1
Western Blot Analysis of SJ GST Protein
2
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were collected following the trypsin digestion, washed with PBS, and lysed with radioimmunoprecipitation assay (RIPA) lysis buffer with phenylmethane sulfonyl fluoride (PMSF). Total protein levels were determined by using BCA Protein Assay Kit (Pierce). The same amount of protein was taken from each sample, the same volume of 2× loading buffer was added, and the sample was cooked in boiling water for 10 min. Proteins were separated on 10% SDS-PAGE, and transferred to polyvinylidene fluoride (PVDF) membranes (Amersham), which were blocked with Tris-buffered saline with Tween 20 (TBST) solution containing 5% skimmed milk for 1 h, and the membranes were incubated with primary antibodies overnight. PVDF membranes were incubated with the secondary antibodies for 2 h, and the results were visualized using the ECL Plus Western Blotting kit (Amersham).
In the signaling pathway analyses, proteins were analyzed by using an automated western blot system (http://www.proteinsimple.com/simon.html ). The following automatic ran was used: separate/immobilize/incubate with primary antibody/wash/ incubate with secondary antibody/wash/ incubate with enzyme substrate/expose [23 (link)–25 (link)].
In the signaling pathway analyses, proteins were analyzed by using an automated western blot system (
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!