NanoBio: Test Induction & Purification: Difference between revisions
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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. | 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=== | ===Materials=== | ||
*Culture* (see procedure for specifics) | *Culture/colony* (see procedure for specifics) | ||
*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) | ||
| Line 15: | Line 15: | ||
*#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. | *#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 in shaker 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. | *Remember to pellet 2mL of uninduced cells too. | ||
| Line 21: | Line 21: | ||
===Extraction=== | ===Extraction=== | ||
#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. | #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. | ||
#Centrifuge 10 minutes at fast. | |||
#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. | ||
#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. | ||
===Run them on a gel=== | ===Run them on a gel=== | ||
See [[NanoBio: Protein Gels|Protein Gels]]. | 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. | *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) | [[User:Arthur K Yu|Arthur K Yu]] 21:31, 22 July 2008 (UTC) | ||
Latest revision as of 15:34, 22 July 2008
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
- 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.
- Centrifuge 10 minutes at fast.
- 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.
- Centrifuge 10 min at fast.
- 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)
