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
  1. Back dilute. Take 40uL saturated culture and add to 2mL LB/antibiotic. Incubate in shaker for 2-3 hours.
  2. Save 1-2mL of saturated culture as uninduced cells.
  3. Add IPTG. Add enough so your final concentration is 1mM. Incubate in shaker for 3-4 hours.
• From a plate
  1. First thing at work, pick a colony and put into LB/antibiotic. Incubate in shaker for 6-8 hours.
  2. Save 1-2mL of your culture so you can run it as uninduced for comparison.
  3. 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.

• 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.

Arthur K Yu 21:31, 22 July 2008 (UTC)