To analyze the presence of hypusine-containing eIF5A, cultures of the yeast strain VZL1074 transformed with plasmids containing the new eIF5A mutants were grown overnight at 25 °C in 5 mL of SC-ura media with raffinose, and then the cells were transferred into SC-ura media containing tetracycline and galactose to express the eIF5A mutants. After 6 hours of induction, samples were lysed in protein lysis buffer A (50 mM Tris-HCl pH 7.5; 50 mM dithiothreitol; 5 mM EDTA; 5% SDS; 5% glycerol; 2x Protease Inhibitor Cocktail), and 15 μg of whole cell extracts (WCE) were resolved by SDS-PAGE and transferred to a nitrocellulose membrane. We proceeded to perform immunoblot analysis using a rabbit polyclonal anti-eIF5A (yeast) antibody at a 1:15,000 dilution (Valentini et al. 2002 (link)), a rabbit polyclonal anti-hypusine antibody (Millipore) at a 1:1,500 dilution (Mandal et al. 2015 (link)) and a rabbit polyclonal anti-RPL5 (yeast) antibody at a 1:15,000 dilution with an enhanced chemiluminescence detection system on a Li-Cor C-Digit Blot Scanner.
Protocol detail
Immunoblot Analysis of eIF5A and Hypusine
Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link:
Access Free Full Text.
Anti antibody
Antibody
Buffer
Cell extracts
Cells
Chemiluminescence
Digit
Dilution
Dithiothreitol
Edta
Eif5a
Galactose
Glycerol
Hypusine
Immunoblot analysis
Membrane
Nitrocellulose
Plasmids
Protease inhibitor
Protein a
Rabbit
Raffinose
Rpl5
Sds page
Strain
Tetracycline
Tris
Yeast
Corresponding Organization :
Other organizations : Universidade Estadual Paulista (Unesp), Universidade Federal de São Carlos, Universidade de São Paulo, University of California, Davis
Top 5 similar protocols
Variable analysis
independent variables
- EIF5A mutants
- Presence of hypusine-containing eIF5A
- Growth conditions (25 °C in 5 mL of SC-ura media with raffinose, then SC-ura media containing tetracycline and galactose)
- Cell lysis (protein lysis buffer A: 50 mM Tris-HCl pH 7.5; 50 mM dithiothreitol; 5 mM EDTA; 5% SDS; 5% glycerol; 2x Protease Inhibitor Cocktail)
- Sample preparation (15 μg of whole cell extracts (WCE) resolved by SDS-PAGE and transferred to a nitrocellulose membrane)
- Rabbit polyclonal anti-eIF5A (yeast) antibody at a 1:15,000 dilution
- Rabbit polyclonal anti-RPL5 (yeast) antibody at a 1:15,000 dilution
- Rabbit polyclonal anti-hypusine antibody (Millipore) at a 1:1,500 dilution
Annotations
Based on most similar protocols
Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.
Sign up for free or login to display all annotations
As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.
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!