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Experiment Workflow, Search protein's amino acid sequence against…
Experiment Workflow
Search protein's amino acid sequence against BlastP
Find similar protein with high percentage of identities
View alignments and highly conserved domains
Extract cells from tissue sample
Add cell lysis buffer to pellet and resuspend for 1 min (leave on ice)
Incubate for 10 mins at room temperature
Sonicate at 10s pulse, 70% amp (rest on ice for 30s)
Repeat step 4 times
Centrifuge
Collect supernatant to a new tube
Add Laemmli buffer to the tube
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Reconstitute a small amount of sample in rehydration buffer and place on IPG strip
Dispense sample along the edge of tray
Place IPG on focusing tray with gel-side up
Place focusing tray into protein IEF cell
Equilibrate and run the gel with 10kv
Remove IPG strip and dry on filter paper
Load DPT buffer in the lane and equilibrate IPG strip
Shake for 15 minutes and discard the buffer
Repeat step with Iodacetamide
Equilibrate and remove IPG strips and rinse with SDS-PAGE running buffer
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