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.
- 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.
- 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.