lab 7 & 8 DGD - BCH 3356 English Thursday...

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BCH 3356 English – Thursday 8:30-10:00 TBT333 English – Friday 2:30-4:00 MNT203 Megan Rose –
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Lab 5 Reinoculation of the positive transformant cell line (selected during lab 4) into a liquid culture and IPTG- induction for overexpression of T7 RNA polymerase. AMP IPTG
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IPTG t=0 No IPTG t=0 No IPTG t=1.5 No plasmid t=0 No plasmid t=1.5 IPTG t=1.5 Lab 5 – expected results
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Lab 6 All of the bacterial proteins are in the supernatant, so the T7 RNA polymerase will be further purified by His-tag affinity chromatography His-tag Affinity chromatography SDS-PAGE Supernatant before after
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Lab 6 - Hypothetical results Flowthrough contains proteins with no affinity for column beads. Washes contain proteins with low affinity for column beads. Eluted fractions contain the recombinant T7 RNA polymerase ***this example uses another protein, NOT RNA polymerase T7*** OS is the sample that was loaded onto the column FT = flowthrough W1 = wash 1 W2 = wash 2 E = elution ( in this example they eluted the protein in different fractions, in the lab you
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Gradient SDS-PAGE proteins migrate from lower to higher concentrations of polyacrylamide This means that the pores at the top of the gel are bigger than the ones at the bottom of the gel. As the gel pores decrease in size, the migration rate of proteins also decreases. It is more difficult to move through very
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Gradient SDS-PAGE o Enabling a broader range of protein sizes to be separated. o the molecules at the front are delayed as they are moving through a higher concentration area while those behind can catch up due to a faster migration through lower acrylamide concentration.
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Gradient 4-15% gel Lan e Sample Sampl e volum e (uL) 2X Loadin g buffer (uL) Loadin g volum e (uL) 1 Rainbow marker (7.5ug/5ul) - - 5 2 Input control, 5 ug or a maximum of 12.5 uL 3 Flowthrough control, 5 ug or a maximum of 12.5 uL 4 Wash1 control, 1 ug or a maximum of 12.5 uL 5 Wash2 control, 1 ug or a maximum of 12.5 uL 6 Elution control, 0.5 ug or a maximum of 12.5 uL 7 Purified protein, 0.5 ug or a maximum of
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lab 7 & 8 DGD - BCH 3356 English Thursday...

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