BMD RES 5HA Lecture Notes - Immunoprecipitation, Protein Tag, Start Codon
4 May 2018
School
Department
Course
Professor
Midterm •
Think of two questions to ask Dr. J ◦
Due on Thursday May 3rd before the Q&A ◦
Dr. J research •
How to determine if two proteins are interacting with each other ◦
Coimmunoprecipitation ‣
Antibodies are needed and tags (made of a couple amino acids ) where the antibodies bind to, then the antibody attaches to ‣
The DNA sequence has to be given to the yeast , then the cells are opened (whole cell lysis) , put into test tube with beads ‣
specific for the antibody
We don’t know if the isoforms are dimerizing ‣
Transcription factors are known to dimerize which is why she did this experiment ◦
‣
She completed the experiment twice with changing the tags to make sure that the tags didn’t interfere with the interaction ◦
You can introduce errors, false interactions due to the tags ‣
This is standard practice ‣
Co-immunoprecipitation •
Make sure to put the protein tag somewhere that is away from the interaction (potential introduce error ) ◦
Two antibodies are use ◦
Pull down (IP) - to attach to the bead ‣
Detection antibody - detected in the western blot ‣
Dr. j data •
Lane 1 and 2 are not introducing nsDNA - TAP ◦
Recap: what we are doing ‣
‣
Her data ◦
Document Summary
Think of two questions to ask dr. j. Due on thursday may 3rd before the q&a. How to determine if two proteins are interacting with each other. Antibodies are needed and tags (made of a couple amino acids ) where the antibodies bind to, then the antibody attaches to. The dna sequence has to be given to the yeast , then the cells are opened (whole cell lysis) , put into test tube with beads speci c for the antibody. We don"t know if the isoforms are dimerizing. Transcription factors are known to dimerize which is why she did this experiment. She completed the experiment twice with changing the tags to make sure that the tags didn"t interfere with the interaction. You can introduce errors, false interactions due to the tags. Make sure to put the protein tag somewhere that is away from the interaction (potential introduce error ) Pull down (ip) - to attach to the bead.
