Oct 11, 2022
Preparation of bacterial cell lysate for SDS-PAGE (2022 iGEM)
- 1Fudan University
Protocol Citation: Team Fudan iGEM 2022. Preparation of bacterial cell lysate for SDS-PAGE (2022 iGEM). protocols.io https://dx.doi.org/10.17504/protocols.io.14egn2zwyg5d/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: Working
We use this protocol and it's working
Created: October 10, 2022
Last Modified: October 11, 2022
Protocol Integer ID: 71103
Abstract
Preparation of bacterial cell lysate for SDS-PAGE
Preparation of bacterial cell lysate for SDS-PAGE
Preparation of bacterial cell lysate for SDS-PAGE
Grow the cells overnight, to an OD600 above 1. If IPTG induction needed, at it around OD600 0.2-0.3 (keep IPTG stock solution at -20 freezer).
Take one 1 ml of OD600 = 1 from each sample. Correct the sample volume to obtain an equivalent size pellet. For us, as quantified using NanoCym950 nanoparticles (equivalent to the size of E. coli), 1 OD600 equals to 10^8 nanoparticles per mL. We estimate each well on SDS-PAGE contains proteins from 2*10^6 bacteria.
16h
Spin down the bacterial cells for 1 min at 13,000 rpm in a microfuge.
10m
Aspirate any trace of supernatant with a vacuum line avoiding touching the pellet, but removing all liquid.
1m
To each cell pellet, add 250 µL of distilled water, resuspend the pellet. Add 250 µL 2x SDS sample buffer (see below). Heat with a 95 degree heating block for 10 minutes. Store the samples at -20 degree freezer until SDS-PAGE.
1m
Receipt
Receipt
2x SDS sample buffer, works for Tris-Cl or MOPS based gels
- Glycerol 20 ml
- SDS 4 g
- 0.5 M Tris-HCl pH 6.8 stock solution 25 ml
- Bromo Phenol Blue 1 mg; dissolve and volume to 95 ml
- add 5% β-Mercaptoethanol before usage