methods

unique happy places

protein has a special feature that sticks to the resin (agarose beads with antibodies or metals) most selective

resin is charged and is attracting charged residues on proteins of interest separation is achieved by changes of pH which releases bound proteins

resin is not charged and is attracting non-charged residues on proteins of interest salt exposes hydrophobic residues of proteins separation is achieved by changing [salt] –> water is now available (water is polar) –>

in resin (beads), there are little channels that allow proteins to flow through big proteins flow through faster (because they avoid channels – small proteins flow through channels) least selective

antibodies

enzyme-linked immunosorbent assay an antibody binds an antigen of interest and produces a detectable signal

part of a large antigen that interacts with the antibody not recognized when protein is unfolded

a single Ab binds the antigen and carries the signal

primary Ab (usually polyclonal) binds the antigen; secondary Ab binds the primary Ab and carries the signal

both Ab bind the antigen at unique epitopes Order of addition: • capture Ab is absorbed to the well • antigen is added • primary Ab is added –> binds to antigen and carries signal

antigen presence is indicated by an absence or decrease of signal “if antigen is present is sample, we lose the signal” “positive result is lack of signal” Order of addition: • antigen is added • primary Ab + sample • secondary Ab

• formaldehyde covalently connects DNA and protein at the location of a non-covalent interaction (cross-link) • the antibody used is specific to a DNA-binding protein • collects all DNA crosslinked to the protein (collects all directly and indirectly linked DNA sequences) • reversing the covalent bonds with formaldehyde releases the DNA • DNA can be analyzed

when blood typing, the interaction of antigen and antibody – linking of blood cells together

RhoGAM (anti-D) to prevent the production of maternal antibodies against fetal Rho/D antigen

HER2+ breast cancers attaches to HER2 receptors preventing signal to cell to divide

DNA RNA Protein SNOW DROP