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Scenario 2 should be answered given gel electrophoresis marks. Please give a guideline on how to answer this question. SCENARIO 1 IS JUST FOR BACKGROUND
Scenario 2 should be answered given gel electrophoresis marks. Please give a guideline on how to answer this question.
SCENARIO 1 IS JUST FOR BACKGROUND INFORMATION
2. Hypothetical protein analysis scenario Scenario 2: Following a purification process, it is routine practice to analyse the purification fractions via SDS-PAGE analysis with visualisation through Coomassie blue staining. On the image given in the answer document, draw the locations of proteins present in the relevant fractions generated by your first purification process (not the CM-cellulose step) i.e., the unbound proteins (where applicable), and the eluted, purified proteins, each drawn in a separate lane. If you only have 2 fractions, you need only fill in lanes 3 and 4. If you have more peaks in your elution profile, you'll need to fill out more lanes. In the image provided, Lane 2 provides an example of how proteins should be annotated in the other lanes. Lane 2 shows the collection of proteins that accompany TMS3 in the original cell-free extract in Scenario 1 (excluding TMS3) before CM-cellulose purification. You must assume that you have treated the protein samples by heating at 100C for 5 mins in a reducing denaturing buffer (60 mM Tris pH 7.5, 2% (w/v) SDS, 0.04% (w/v) bromophenol blue, 10% (v/v) glycerol, 100 mM DTT) prior to separation through a 420% (w/v) gradient polyacrylamide gel in Tris-glycine SDS-PAGE buffer (25mM Tris pH 8.4, 192mm glycine, 0.1% (w/v) SDS). Assume that visualisation of proteins was achieved by Coomassie Blue staining. Use arrows to label the proteins with their names in both Lane 2 and your additional completed lanes, and complete the figure legend with the description of your additions to the gel image. 1 2 3 4 5 6 kDa 200 116.3 97.4 66.21 I III TILL || 45.5 31.5 21.5. 14.4 6.5 Lane 1: MW protein marker Lane 2: Proteins in cell-free extract (excl. TMS3) 1. Hypothetical protein purification scenario Scenario 1: Your task is to provide the rationale, methodology and expected outcomes for a purification experiment. You are expected to further purify an overexpressed protein, TMS3, from a mixture of proteins (additional proteins are shown in Table A below). Here is a little bit of information about the desired protein, TMS3: TMS3 has a pl of 7.0 and a subunit molecular mass of 25.9 kDa TMS3 was over-expressed as a fusion protein in a bacterial expression system in E. coli BL21 Rosetta (DE3) cells from the following plasmid: PT7/Lac O/RBS fiori GST Asci PEX-N-GST Vector 5.2 kb TEV Sof! MCS Stop Rsr 11 Mlu T7 terminator ColE1 Lac! TMS3 - in the cell-free extract - was subjected to an initial purification step using CM-cellulose batch purification. Proteins were bound to 200 uL resin in a buffer of 20 mM sodium phosphate pH 6.8, 1 mM EDTA, washed twice with 5 mL 20 mM sodium phosphate pH 6.8, 1 mM EDTA buffer and eluted by a single-step elution in 3 mL 20 mM sodium phosphate, pH 8.5, 0.5 M NaCl, 1 mM EDTA. The supernatant was transferred to an Ultra15A device (10kDa MWCO) and concentrated by ultrafiltration down to a 0.5-mL sample. The sample was stored at 4C in preparation for the next steps of purification. Table A. Additional proteins contained in the cell-free extract subjected to CM-cellulose purification Protein name pl Protein 'number M, (kDa) 1 AD1 78 7.6 2 Hyp2 98 7.0 3 HUG1 52 7.1 4 GI5-GST fusion 126 7.1 5 Hyp1 108 6.2 6 ATMS 9.5 7.5 7 Hyp-th2 25* 8,6 Note 1: Hyp-th2 protein is a tetrameric native protein, subunit mw given* Note 2: the cell-free extract also contains TMS3 Materials provided: 0.5 mL sample of partially purified proteins containing TMS3 O o Buffers for use: 20 mM sodium phosphate, pH 7.2,1 mM EDTA 20 mM sodium phosphate, pH 7.2, 100 mM NaCl, 1 mM EDTA 20 mM sodium phosphate, pH 7.2, 0.75 M NaCl, 1 mM EDTA 50 mM Tris.HCI, pH 6.5, 1 mM EDTA 50 mM Tris.HCl, pH 6.5, 1 mM EDTA, 25 mM imidazole 50 mM TBS, PH 7.5, 100 mM NaCl, 1 mM EDTA, 1mM DTT, 50 mM reduced glutathione 50 mM TBS, pH 7.5, 100 mM NaCl, 1 mM EDTA, 1mM DTT o O o o o o o Purification materials: Amicon Ultra 15A Ultrafiltration device, MWCO 10,000 Amicon Ultra15B Ultrafiltration device, MWCO 30,000 DEAE-cellulose column, (pK11.5), stored in 20 mM sodium phosphate, pH 7.2, 1 mM EDTA Immobilised glutathione agarose resin column stored in 50 mM TBS, PH 7.5, 100 mM NaCl, 1 mM EDTA, 1mM DTT Ni-NTA resin column stored in dH20 Sephadex G-100 column stored in dH 0 Sephadex G-75 column stored in dH20 o o o 2. Hypothetical protein analysis scenario Scenario 2: Following a purification process, it is routine practice to analyse the purification fractions via SDS-PAGE analysis with visualisation through Coomassie blue staining. On the image given in the answer document, draw the locations of proteins present in the relevant fractions generated by your first purification process (not the CM-cellulose step) i.e., the unbound proteins (where applicable), and the eluted, purified proteins, each drawn in a separate lane. If you only have 2 fractions, you need only fill in lanes 3 and 4. If you have more peaks in your elution profile, you'll need to fill out more lanes. In the image provided, Lane 2 provides an example of how proteins should be annotated in the other lanes. Lane 2 shows the collection of proteins that accompany TMS3 in the original cell-free extract in Scenario 1 (excluding TMS3) before CM-cellulose purification. You must assume that you have treated the protein samples by heating at 100C for 5 mins in a reducing denaturing buffer (60 mM Tris pH 7.5, 2% (w/v) SDS, 0.04% (w/v) bromophenol blue, 10% (v/v) glycerol, 100 mM DTT) prior to separation through a 420% (w/v) gradient polyacrylamide gel in Tris-glycine SDS-PAGE buffer (25mM Tris pH 8.4, 192mm glycine, 0.1% (w/v) SDS). Assume that visualisation of proteins was achieved by Coomassie Blue staining. Use arrows to label the proteins with their names in both Lane 2 and your additional completed lanes, and complete the figure legend with the description of your additions to the gel image. 1 2 3 4 5 6 kDa 200 116.3 97.4 66.21 I III TILL || 45.5 31.5 21.5. 14.4 6.5 Lane 1: MW protein marker Lane 2: Proteins in cell-free extract (excl. TMS3) 1. Hypothetical protein purification scenario Scenario 1: Your task is to provide the rationale, methodology and expected outcomes for a purification experiment. You are expected to further purify an overexpressed protein, TMS3, from a mixture of proteins (additional proteins are shown in Table A below). Here is a little bit of information about the desired protein, TMS3: TMS3 has a pl of 7.0 and a subunit molecular mass of 25.9 kDa TMS3 was over-expressed as a fusion protein in a bacterial expression system in E. coli BL21 Rosetta (DE3) cells from the following plasmid: PT7/Lac O/RBS fiori GST Asci PEX-N-GST Vector 5.2 kb TEV Sof! MCS Stop Rsr 11 Mlu T7 terminator ColE1 Lac! TMS3 - in the cell-free extract - was subjected to an initial purification step using CM-cellulose batch purification. Proteins were bound to 200 uL resin in a buffer of 20 mM sodium phosphate pH 6.8, 1 mM EDTA, washed twice with 5 mL 20 mM sodium phosphate pH 6.8, 1 mM EDTA buffer and eluted by a single-step elution in 3 mL 20 mM sodium phosphate, pH 8.5, 0.5 M NaCl, 1 mM EDTA. The supernatant was transferred to an Ultra15A device (10kDa MWCO) and concentrated by ultrafiltration down to a 0.5-mL sample. The sample was stored at 4C in preparation for the next steps of purification. Table A. Additional proteins contained in the cell-free extract subjected to CM-cellulose purification Protein name pl Protein 'number M, (kDa) 1 AD1 78 7.6 2 Hyp2 98 7.0 3 HUG1 52 7.1 4 GI5-GST fusion 126 7.1 5 Hyp1 108 6.2 6 ATMS 9.5 7.5 7 Hyp-th2 25* 8,6 Note 1: Hyp-th2 protein is a tetrameric native protein, subunit mw given* Note 2: the cell-free extract also contains TMS3 Materials provided: 0.5 mL sample of partially purified proteins containing TMS3 O o Buffers for use: 20 mM sodium phosphate, pH 7.2,1 mM EDTA 20 mM sodium phosphate, pH 7.2, 100 mM NaCl, 1 mM EDTA 20 mM sodium phosphate, pH 7.2, 0.75 M NaCl, 1 mM EDTA 50 mM Tris.HCI, pH 6.5, 1 mM EDTA 50 mM Tris.HCl, pH 6.5, 1 mM EDTA, 25 mM imidazole 50 mM TBS, PH 7.5, 100 mM NaCl, 1 mM EDTA, 1mM DTT, 50 mM reduced glutathione 50 mM TBS, pH 7.5, 100 mM NaCl, 1 mM EDTA, 1mM DTT o O o o o o o Purification materials: Amicon Ultra 15A Ultrafiltration device, MWCO 10,000 Amicon Ultra15B Ultrafiltration device, MWCO 30,000 DEAE-cellulose column, (pK11.5), stored in 20 mM sodium phosphate, pH 7.2, 1 mM EDTA Immobilised glutathione agarose resin column stored in 50 mM TBS, PH 7.5, 100 mM NaCl, 1 mM EDTA, 1mM DTT Ni-NTA resin column stored in dH20 Sephadex G-100 column stored in dH 0 Sephadex G-75 column stored in dH20 o o oStep by Step Solution
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