Difference between revisions of "Team:UCLA/Notebook/Recombinant Expression/8 July 2015"
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Using the collected supernatant form Sample 1, we optimize and troubleshoot a protocol to purify our protein using nickel column chromatography and gravity filtration. The column contains a Histidine resin that selectively binds to His-tagged proteins. The column is equilibrated and charged before the sample is run through. The sample is diluted in 1X binding buffer in a 1 to 1 volume ratio. The idea is to add solutions of successively higher concentrations of imidazole. Imidazole has an extremely high affinity for the nickel charged column, and by the time the elution buffer is added (250mM), imidazole will outcompete our Tamura proteins in binding, allowing the Tamura proteins to elute and drain through the column to be collected as the final eluate. | Using the collected supernatant form Sample 1, we optimize and troubleshoot a protocol to purify our protein using nickel column chromatography and gravity filtration. The column contains a Histidine resin that selectively binds to His-tagged proteins. The column is equilibrated and charged before the sample is run through. The sample is diluted in 1X binding buffer in a 1 to 1 volume ratio. The idea is to add solutions of successively higher concentrations of imidazole. Imidazole has an extremely high affinity for the nickel charged column, and by the time the elution buffer is added (250mM), imidazole will outcompete our Tamura proteins in binding, allowing the Tamura proteins to elute and drain through the column to be collected as the final eluate. | ||
| − | PROBLEM: The first step of the protocol states to run the sample diluted in 1X binding buffer through the column and collect what drips through as Fraction 1. About 5mL of Fraction 1 was collected before the column became clogged, preventing further use of gravity filtration. The contents of Fraction 1 are expected to be any unbound proteins, a.k.a. contaminant proteins that are not Tamura. As a result, we hypothesize that blockage is likely due to these contaminant proteins | + | PROBLEM: The first step of the protocol states to run the sample diluted in 1X binding buffer through the column and collect what drips through as Fraction 1. About 5mL of Fraction 1 was collected before the column became clogged, preventing further use of gravity filtration. The contents of Fraction 1 are expected to be any unbound proteins, a.k.a. contaminant proteins that are not Tamura. As a result, we hypothesize that blockage is likely due to these contaminant proteins being too big in size, unable to flow through our smaller-sized pores in the column. Tomorrow, we will troubleshoot the protocol or attempt a new method to purify our protein sample. |
Latest revision as of 20:11, 13 July 2015
Using the collected supernatant form Sample 1, we optimize and troubleshoot a protocol to purify our protein using nickel column chromatography and gravity filtration. The column contains a Histidine resin that selectively binds to His-tagged proteins. The column is equilibrated and charged before the sample is run through. The sample is diluted in 1X binding buffer in a 1 to 1 volume ratio. The idea is to add solutions of successively higher concentrations of imidazole. Imidazole has an extremely high affinity for the nickel charged column, and by the time the elution buffer is added (250mM), imidazole will outcompete our Tamura proteins in binding, allowing the Tamura proteins to elute and drain through the column to be collected as the final eluate.
PROBLEM: The first step of the protocol states to run the sample diluted in 1X binding buffer through the column and collect what drips through as Fraction 1. About 5mL of Fraction 1 was collected before the column became clogged, preventing further use of gravity filtration. The contents of Fraction 1 are expected to be any unbound proteins, a.k.a. contaminant proteins that are not Tamura. As a result, we hypothesize that blockage is likely due to these contaminant proteins being too big in size, unable to flow through our smaller-sized pores in the column. Tomorrow, we will troubleshoot the protocol or attempt a new method to purify our protein sample.