NanoBio: Test Induction & Purification

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(Extraction)
Current revision (18:34, 22 July 2008) (view source)
(Extraction)
 
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#Remove supernatents and keep them. This is the '''soluble''' portion.
#Remove supernatents and keep them. This is the '''soluble''' portion.
#To the pellet, Add 10mg/mL lysozyme in B-PER and pipet up and down and do a 1 min vortex.
#To the pellet, Add 10mg/mL lysozyme in B-PER and pipet up and down and do a 1 min vortex.
 +
#Centrifuge 10 min at fast.
#Remove supernatents and keep them. This is the '''insoluble''' portion.
#Remove supernatents and keep them. This is the '''insoluble''' portion.

Current revision

Contents

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)

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