NanoBio: Test Induction & Purification

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(New page: ===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 t...)
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*LB w/ appropriate antibiotic
*LB w/ appropriate antibiotic
*IPTG 0.1M or 1M (or your appropriate inducer)
*IPTG 0.1M or 1M (or your appropriate inducer)
-
*B-PER protein extraction solution
+
*B-PER protein extraction reagent
 +
*Lysozyme at 10 mg per mL of B-PER (for insoluble extraction)
===Growing up culture===
===Growing up culture===
*'''From overnight culture'''
*'''From overnight culture'''
*#Back dilute. Take 40uL saturated culture and add to 2mL LB/antibiotic. Incubate in shaker for 2-3 hours.
*#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.
*#Add IPTG. Add enough so your final concentration is 1mM. Incubate in shaker for 3-4 hours.
*'''From a plate'''
*'''From a plate'''
*#First thing at work, pick a colony and put into LB/antibiotic. Incubate in shaker for 6-8 hours.
*#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 overnight.
*#Add IPTG. Add enough so your final concentration is 0.25mM. Incubate overnight.
*'''Pellet your cells.''' Suggested 5 min at 16G.
*'''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.
*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===
===Extraction===
-
asdf
+
#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.
-
[[User:Arthur K Yu|Arthur K Yu]] 19:54, 22 July 2008 (UTC)
+
#Remove supernatents and keep them. This is the '''soluble''' portion.
 +
#Re-pellet samples.
 +
#Add 10mg/mL lysozyme in B-PER and pipet up and down and do a 1 min vortex.
 +
#Remove supernatents and keep them. This is the '''insoluble''' portion.
 +
===Run them on a gel===
 +
See [[NanoBio: Protein Gels|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.
 +
[[User:Arthur K Yu|Arthur K Yu]] 21:31, 22 July 2008 (UTC)

Revision as of 17:31, 22 July 2008

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* (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 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. Remove supernatents and keep them. This is the soluble portion.
  3. Re-pellet samples.
  4. Add 10mg/mL lysozyme in B-PER and pipet up and down and do a 1 min vortex.
  5. 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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