L8 Western F19 student.pptx - Your Combined Lab Report is due BIOL3380 Fall 2019 The Big Picture Experiment 8 –Western Blot Analysis of rGFP fractions

L8 Western F19 student.pptx - Your Combined Lab Report is...

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Your Combined Lab Report is due_______________________ BIOL3380 - Fall 2019
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The Big Picture Experiment 8 –Western Blot Analysis of rGFP fractions BIOL3380 - Fall 2019
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Experiments #3-8 The overall goal/purpose of this series of experiments is to see if we can express and purify a His 6 -tagged recombinant form of GFP (rGFP) from the E. coli strain BL21(DE3)<pLysS><pRSETA-GFP UV > using the Ni+2 agarose affinity chromatography technology. This week’s experiment will allow us to: BIOL3380 - Fall 2019
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Express rGFP in E. coli Grow the E. coli and induce (turn on) the expression (transcription/translation) of rGFP Grow 3 hours Induce expression with IPTG Harvest 15ml G0 Sample G3 Sample centrifuge G3-15ml pellet G0 = G culture before induction G3 = G culture 3 hours post induction decant supernatant BIOL3380 - Fall 2019
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G3-15ml pellet isolate a crude extract GCE purify His6 tagged proteins Collect and store samples for further analysis Experiment #4 - Purification of rGFP using Ni +2 agarose GCE GCE = rG FP C rude E xtract W = wash fraction E = elution fraction freeze/thaw method Ni +2 affinity chromatography Develop column with a wash buffer followed by an elution buffer Washes Elutions W2/3 slight fluorescence E2/3 strong fluorescence E1 little to no fluorescence W1 W6 E1 E6 BIOL3380 - Fall 2019
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Experiment #5 Determining Protein concentration of rGFP fractions W1-W6 E1-E6 Bradford Assay Using a standard curve we can estimate the in a sample by off the standard curve. total protein amount protein concentration protein (ug) Abs (595nm) Knowing the volume of the sample we can calculate the Create a BSA standard curve interpolation BIOL3380 - Fall 2019
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BIOL3380 - Fall 2019 Experiment #6 SDS-PAGE analysis of rGFP fractions GCE W2-W3* E2-E3* Purpose G0 G3 Imaging Estimate protein purity and relative MW *Use the 2 samples with highest fluorescence
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Experiment #7 SDS-PAGE/Western Transfer GCE W2-W3 E2-E3 G0 G3 SDS-PAGE Transfer Nitrocellulose membrane (blot) *Use the 2 fractions with highest fluorescence BIOL3380 - Fall 2019
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BIOL3380 - Fall 2019 rGFP kD rGFP His 6 Xpress 150 250 100 75 37 25 MW(kD) 50 20 15 Estimated MW His 6 Xpress degradation product of rGFP: Terminus Terminus Contaminant
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Experiment #8 Western Blot Membrane Membrane BIOL3380 - Fall 2019
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Detection methods of proteins in a SDS-PAGE gel BIOL3380 - Fall 2019
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