Slide 1THIS WEEKHow do we view our PCR results?Agarose Gel ElectrophoresisAgaroseGel ElectrophoresisAgarose Gel ElectrophoresisNEXTSlide 9We will perform two experiments:Protein ElectrophoresisPAGE ApparatusDenaturing proteinsProtein GelGelatin ZymographyGelatin Zymography, cont.Next week’s ProtocolChapter 6THIS WEEK•REVIEW:1. Isolate RNA2. Reverse Transcription of RNA into cDNA3. PCR to amplify MMP-9 (what we’re testing for), and β-actin (control)•I did this for you already4. View results using Agarose Gel Electrophoresis•What you’re doing today!*We are viewing RNA, thus transcriptional changes!How do we view our PCR results?Gel ElectrophoresisAgarose Gel Electrophoresis•Method of separating molecules on the basis of charge and/or size •Samples subjected to an applied electric field move through the gel according to their charge to mass ratio•Ethidium bromide binds to double-stranded DNA and fluoresces red-orange under UV light.•We always describe the DNA we in the gel in ‘base pairs’.AgaroseGels made of Agarose are used to separate Nucleic AcidsGel Electrophoresis1. Melt agarose in buffer and pour into gel cast; place combs to create wells and let cool2. Mix DNA sample with loading dye (colored dye with glycerol to make samples dense so they don’t float out of the well.)3. Load samples into wells4. Apply charge so DNA will move through gelDNA is negatively charged due to phosphate backbone5. Visualize using Ethidium bromide under UV lightAgarose Gel Electrophoresis•Obtain new 1.5mL Eppendorf tube.•Be sure to note your group number on the PCR tubes!•Add 15uL of your PCR product with MMP9 primers. •Add 15uL of your PCR product with β-actin primers•Add 8uL of loading dye•Load 20uL into gel:–I will load 100 basepair ladder in lanes 1 •DO NOT LEAVE!!!NEXT•We’ve used May-Grünwald/Giemsa staining to view:MORPHOLOGICAL CHANGES•We’ve used RT-PCR to view:TRANSCRIPTIONAL CHANGES•Now we want to view:TRANSLATIONAL CHANGES AND ENZYMATIC ACTIVITYPMA Signal Transduction PathwayIn the presence of PMA a signal is triggered via receptors in the cell membrane to a secondary messenger, PKC-β which in turn induces a response in the DNA for the cell to modify and produce the end product, MMP-9Experimental focus:Protein expressionWe will perform two experiments:1. SDS-PAGE:1. This is a form of gel electrophoresis that will show us total protein content2. Zymogram analysis:1. A type of SDS-PAGE that will allow us to detect enzymatic activity of MMP-9.Protein Electrophoresis•Polyacrylamide Gel Electrophoresis (not agarose)This type of gel provides better resolution for proteins…but the principle is still the same: proteins are separated based on their charge and size!PAGE Apparatushttp://web.chemistry.gatech.edu/~williams/bCourse_Information/4581/techniques/gel_elect/gel.jpgDenaturing proteins•Proteins are denatured first so that they will all travel towards the positive electrode•Samples are typically heated prior to running them on the gel so that shape will not affect their migration rate•Components of the gel and loading buffer also denature the proteins: SDS (sodium dodecyl sulfate)•This molecule is an anionic and amphipathic detergent, meaning that it is negatively charged and contains both hydrophobic and hydrophillic regions.•These characteristics allow SDS to interact with all parts of the protein, allowing the protein as a whole to take on the negative charge of SDSProtein Gel •Coomassie Blue binds to the peptide bonds of the proteins allowing them to be seen as a blue band on the polyacrylamide gel.•Destaining removes any residual Coomassie Blue from the gel and not the dye from the proteins.•Visualize blue bands on the clear gel background. •Remember, the gel is showing TOTAL protein isolation from treated HL-60 cells.Gelatin Zymography•A type of protein electrophoresisPolyacrylamide gels are made with a gelatin in the gel mixture•This gelatin is the substrate for Gelatinase enzymes (recall that MMP-9 is a Gelatinase)•The samples loaded onto the gel are NOT heated but ARE loaded with SDS–After running the gel, the entire gel is soaked in a solution which allows the enzymes to renature–The entire gel is incubated overnight in a solution which allows the enzymes to act on their substrateGelatin Zymography, cont.•After the overnight incubation, the gel is stained in Coomassie Brilliant Blue. The areas where the Gelatinases digested away the gelatin appear as clear bands on a blue background. –The lighter the band, the more enzymatic activity took place at that location.MMP-9: 92 kDaMMP-2: 72 kDaNext week’s Protocol•Keep in mind we are using two different tests to evaluate HL-60 cellular protein presence and function:1. SDS-PAGE – to demonstrate protein presence2. Zymography – to evaluate protein functionBoth protocols utilize gel electrophoresis methodology with the main difference being in the formulation of the gel.•We will be loading growth medium (cell spin supernatant) onto our zymograms instead of HL-60 protein extracts.•MMP-9 is cleaved outside of the cell in order to produce the active enzyme.•REMEMBER: For MMP-9 to become active it is secreted from the cell.•CONTROL FOR EXPERIMENT: MMP2 (expressed in both PMA-treated HL-60 cells (monocytes) & DMSO-treated HL-60 cells