NanoBio: Test Induction & Purification

Overview
After you have verified your construct, you need to make sure your plasmid yields the right protein. You are going to do a small scale crude protein extraction and compare the uninduced cells VS induced soluble part of cells VS induced insoluble part of cells. Ideally, a lot of the right protein is in the induced soluble part.

Materials

 * Culture/colony* (see procedure for specifics)
 * LB w/ appropriate antibiotic
 * IPTG 0.1M or 1M (or your appropriate inducer)
 * B-PER protein extraction reagent
 * Lysozyme at 10 mg per mL of B-PER (for insoluble extraction)

Growing up culture

 * From overnight culture
 * Back dilute. Take 40uL saturated culture and add to 2mL LB/antibiotic. Incubate in shaker for 2-3 hours.
 * Save 1-2mL of saturated culture as uninduced cells.
 * Add IPTG. Add enough so your final concentration is 1mM. Incubate in shaker for 3-4 hours.
 * From a plate
 * First thing at work, pick a colony and put into LB/antibiotic. Incubate in shaker for 6-8 hours.
 * Save 1-2mL of your culture so you can run it as uninduced for comparison.
 * Add IPTG. Add enough so your final concentration is 0.25mM. Incubate in shaker overnight.
 * Pellet your cells. Suggested 5 min at 16G.
 * Remember to pellet 2mL of uninduced cells too.
 * You may save your pellet at 4 C for use tomorrow or -20C if you're going to wait longer. Freezing will cause crystallization but it doesn't matter because this is just a crude analytical purification.

Extraction

 * 1) Add B-PER to your pellets. 300uL BPER for the pellet from 2mL culture is good. Pipet up and down and vortex for 1 minute.
 * 2) Centrifuge 10 minutes at fast.
 * 3) Remove supernatents and keep them. This is the soluble portion.
 * 4) To the pellet, Add 10mg/mL lysozyme in B-PER and pipet up and down and do a 1 min vortex.
 * 5) Centrifuge 10 min at fast.
 * 6) Remove supernatents and keep them. This is the insoluble portion.

Run them on a gel
See Protein Gels. Arthur K Yu 21:31, 22 July 2008 (UTC)
 * Make sure you have the molecular weight of your protein handy so you know what gel composition to use, and so you know where your band is.