Note Jan 30, 2013 lecture 2

Immune system creates more than 1 ab against same

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Immune system creates more than 1 Ab against same antigen giving you a polyclonal- more than one Ab mixed together Isolate a single B lymphocyte makes anti BRAF Ab Fused to immortalized B cell Hybridoma immortal and produces monoclonal Wheres BRAF? Cll fractionation Lyse cells Series of spinning Whole cells, nucleus, large microsomes are in the bottom Spin supernatant - mitochondria, lysosomes Keep spinning to retrieve different cell fractions western/immunoblot Run on SDS-Page gel Stain with coomassie blue Transfer to protein nitrocellulose paper BRAF is found in the cytoskeleton and the lysate- everything and a more specific location
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BRAF IS 84KDa- where we should see a band Microscopy/immuno staining/immunohistochemistry Chemically freeze the cells-fixation Add formaldehyde-cross links proteins to one another Permeabilize the membranes Add primary antibody- binds anywhere the BRAF is Secondary antibody binds to the primary Ab Should see a signal throughout the cytosol There should be a blank spot where the nucleus is BRAF is in the cytosol How does BRAF function? What is BRAF acting on? Colocalization Is another candidate protein in same pace at same time as BRAF? BRAF found in thyroid cancer Candidate protein MST1- also found mutated in thyroid cancer Wheres MST1? Cell fractionation, western Immunostain for MST1 What if we don't have antiMST1 antibody? Alter gene to have an antibody that binds to MST1 Epitope is an AA sequence that you can add Ex:GST DYKDDDDK Does MST1 bind BRAF? Test tube experiment Add beads that bind to BRAF Spin beads down and the pellet contains BRAF and anything that binds to it Is MDT1 in the supernatant or the pellet?
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