Several experiments and a specific control for each are outlined below along with the outcome of that experimental control. You need to explain what each control tests and what you can to address the problem you have diagnosed. Experiment Control Transformation of E. coli with a ligation mix of a vector cut with two different enzymes and insert Pull-down assay using GST-tagged protein bound to glutathione column Immunolocalization -detect subcellular localization of a specific protein in sections of Drosophila brains RT-PCR Yeast two-hybrid testing of two putative interactors. Promoter fusion of a liver-expressed gene Transform the E. coli strain with the ligation mix of the double cut vector alone (i.e., no insert added) Evaluate binding of extract proteins to an 'empty' glutathione column (no epitope tagged protein added) Do the experiment. with sections of brain from a mutant that does not make the target protein- Carry out the RT-PCR, leaving out the reverse transcriptase Test for expression of the bait and prey proteins in the yeast strain by immunoblotting- should see protein expression of expected size Transform and look for liver-specific expression What is the control testing? Tests whether the vector is completely cut Tests the specificity of the primary antibody for the target Tests if the bait and prey fusion proteins are are intact (not proteolytically degraded by the host) Tests that you have the full promoter Example results of the control test You see colonies on the plate for this transformation You see evidence of protein binding to the empty column You see binding in the sections that do not express the target protein You see genomic DNA contamination The cultures you tested show evidence of protein proteolytic cleavage You see expression that is not completely liver- specific Given the control results outlined at left what can you do to fix this? Ignore these proteins if they are identified in your test samples Design primers that flank an intron for the PCR step or re-isolate the RNA free of genomic DNA