Sep 17, 2024

Public workspaceSmall Scale IPTG Induction | Protein Expression

DOI
dx.doi.org/10.17504/protocols.io.yxmvme47og3p/v1
  • 1University of Washington
Catherine M Gohar
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DOI: dx.doi.org/10.17504/protocols.io.yxmvme47og3p/v1
Protocol Citation: Catherine M Gohar 2024. Small Scale IPTG Induction | Protein Expression. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvme47og3p/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
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Created: September 17, 2024
Last Modified: September 17, 2024
Protocol Integer ID: 107736
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Abstract
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Before start
It's best to start small-scale protein purification on a Monday or Tuesday. This protocol will take at least 3 days before the first freezing step.
Seed Culture
Seed Culture
1d
1d
Preheat the shaking tube incubator to Temperature37 °C

Label a Amount15 mL culture tube with the vector strain you will be using, and fill with Amount5 mL of LB broth with 1x Kanamycin antibiotic or 5ul of 1000x.

Place the labeled tube into the shaking incubator to warm, then grab an ice bucket and fill with ice.
Take the BL21 glycerol stock from the Temperature-80 °C freezer and place immediately into the ice bucket.

Use a Amount200 µL pipette tip to scrape some of the ice from the tube, and place the tip directly into the culture tube with LB.

Let the culture shake at Temperature37 °C overnight to be completely saturated by the next morning.

12h
Culture Expansion
Culture Expansion
2h
2h
Grab a sterilized Amount250 mL Erlenmeyer flask and fill with 45mL of Terrific Broth and 1x Kanamycin antibiotic.

Prewarm the flask in the shaking tube incubator at Temperature37 °C

Add all Amount5 mL of the seed culture to the Erlenmeyer flask. The culture should expand 1:10 in 1-2 hours, to an OD around 0.4-0.6.

1h 30m
Thaw a stock of IPTG on ice about 30 minutes before you suspect the culture will be at 0.4 OD.
30m
Quantify the OD of your Amount50 mL flask using Amount500 µL of culture in a spectrometer cuvette. Be sure to blank with Amount500 µL of Terrific Broth.

Once OD hits 0.4-0.6, preheat the fridge shaking incubator to Temperature16 °C

DiluteConcentration1 Molarity (M) IPTG stock to the appropriate concentration for protein induction by adding to expanded culture. This will most likely be a range of Concentration100 micromolar (µM) to Concentration1 millimolar (mM)

To calculate IPTG concentration, use V1 x C1 = V2 x C2, where column E is equivalent to V2.

ABCDE
50mL1mM?1000mM50uL
50mL0.8mM?1000mM40uL
50mL0.6mM?1000mM30uL
50mL0.4mM?1000mM20uL
50mL0.2mM?1000mM10uL

Take your IPTG-induced culture and shake overnight at Temperature16 °C for about 16-18 hours.
18h
Pellet Cells
Pellet Cells
5m
5m
Take each Amount50 mL culture and pour into a Amount50 mL conical tube, labelled.

Take the cells to a large or spinning-bucket centrifuge and spin at Shaker500 x g, 4°C, 00:05:00

5m
Decant cells into a bleach waste bin labeled for culture, and dry on a paper towel for 5-10 seconds
10s
Freeze the pellet at Temperature-80 °C by storing in a freezer for storage, or continue to the protocol for cell lysis.