Oct 26, 2019
Version 3
Magnetic Beads Cell Separation V.3
- 1AEGIS - Madrid iGEM 2019
- AEGIS - Madrid iGEM 2019
Protocol Citation: Laura Sánchez, Claudia Troncone Clemente 2019. Magnetic Beads Cell Separation. protocols.io https://dx.doi.org/10.17504/protocols.io.8schwaw
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
We are still developing and optimizing this protocol. If you try it and find a better way to perform it, let us know!
Created: October 26, 2019
Last Modified: October 26, 2019
Protocol Integer ID: 29220
Keywords: magnetic beads, beads, cell separation, aptamers, affinity, separation
Abstract
The aim of this protocol is to separate the aptamers that bind to the cells from the ones that don't. The cells used have a histidin tag that attaches to the beads' streptavidin tag. The beads will then pull the cells and the complex will precipitate. Since the aptamers have high specifity and affinity for particular sequences, those able to recognice the cells will bind to them, being part of the complex. The aptamers that don't recognice the cells, won't bind. This protocol enables to separate aptamers based on their affinity using the target cells.
The magnetic beads from Promega have proven to be an efficient method to separate proteins. With this variation, we aimed to do a proof of concept and test if we could separate cells efficiently as well. Unfortunately, the method didn't work for us and we weren't able to achieve a good result. We still want to optimize this process, but the protocol that we already developed might help other scientists.
Guidelines
Calculations
- Co = initial
- Cb = bound
- Cnb = non bound
- Cf = final
- Cne = non eluted
Cb = C𝑜 − Cnb
𝐶ne = Cb− 𝐶𝑓
Cnb total = Cnb + 𝐶w1 + 𝐶w2
Optimization
Several tests must be performed following the protocol with different values for each parameter, as shown in the next table. The aim is to find the minimum values which result in an acceptable efficiency.
Materials
MATERIALS
High Capacity Magne™ Streptavidin BeadsPromegaCatalog #V7820
Laboratory equipment:
- Magnetic rack
- Micropipettes ( 10-1000 μL)
- Autoclave
- Incubator
- Culture plates
- Eppendorf tubes / microcentrifuge tubes
Reagents:
- PBS
- MagneHis™ Protein Purification System (as indicated above)
- Wash Buffer: HEPES + Imidazole 10%.
- Elution Buffer: HEPES + Imidazole 500 mM.
- Agar
Safety warnings
Lab coat and disposable gloves are recommended throughout the whole procedure.
Before start
Be sure to have enough amount of the buffers as well as the other reagents.
Separation with the MagneHis+
Separation with the MagneHis+
Prepare the magnetic module: remove the magnetic plate and load the necessary number of 1.5-mL microcentrifuge tubes.
Prepare fresh cultured E. coli samples with cell concentrations of 106 CFU/mL in PBS:
Measure the optical density of the bacterial inoculum at 600 nm absorbance (OD 600)
Dilute it until you get the correct concentrations based on the relation: 1 OD < >1E9 CFU/mL [1].
Collect sample Co
Add 1 mL of each sample [2] to a new eppendorf. Add 300 μL of MagneHis™ [3] to the bacteria mixture. Make sure cells are resuspended properly by gentle mixing.
Incubate at room temperature for 30, 45 and 60 min each under gentle continuous agitation to prevent the beads from settling.
Insert the magnetic plate into the magnetic module. Allow the tube to stand for 3 min for maximum recovery.
Remove the supernatant with the pipette, very carefully, and put it in a new tube.
Collect sample Cnb
Add 1 mL of Wash Buffer (or PBS) and resuspend by inversion. Allow the tube to stand for 3 min into the magnetic module.
Repeat the washing process (steps 6-7) twice.
Collect sample Cw1, Cw2
Resuspend the MagneHis™ complex in 100 μL of Elution Buffer. Mix briefly by inversion. Allow the tube to stand for 1’5 min into the magnetic module.
Take the supernatant with the pipette carefully and add it to a 900 μL LB eppendorf.
* Imidazole is very aggressive to the cell membrane, but it is needed to break the interaction between histidine and MagneHis™, therefore a high dilution is performed to stop its effect.
Collect sample Cf
Resuspend the beads in 100 μL LB.
Collect sample Cne
For spread plating, a 10-fold dilution series (until dilution 10-7) is prepared. Plate 100 μLfrom dilutions 10-1, 10-2 and 10-3 onto 3 different agar plates with the pertinent antibiotic. The plates are incubated at 37°C for 48 h. The colonies are numerated, and the result is expressed as CFU/mL [4].
REFERENCES
REFERENCES
We have developed this protocol from reading the following bibliography:
[1] Park J, Gasparrini A, Reck M, Symister C, Elliott J, Vogel J et al. Plasticity, dynamics, and inhibition of emerging tetracycline resistance enzymes. Nature Chemical Biology. 2017;13(7):730-736. doi: 10.1038/nchembio.2376
[2] Lim M, Lee G, Huynh D, Hong C, Park S, Jung J et al. Biological preparation of highly effective immunomagnetic beads for the separation, concentration, and detection of pathogenic bacteria in milk. Colloids and Surfaces B: Biointerfaces. 2016;145:854-861.
[3] http://t-takaya.net/manual/Miltenyi_Biotec_130-090-485.pdf.
[4] Walcher G, Stessl B, Wagner M, Eichenseher F, Loessner M, Hein I. Evaluation of Paramagnetic Beads Coated with Recombinant Listeria Phage Endolysin–Derived Cell-Wall-Binding Domain Proteins for Separation of Listeria monocytogenes from Raw Milk in Combination with Culture-Based and Real-Time Polymerase Chain Reaction–Based Quantification. Foodborne Pathogens and Disease. 2010;7(9):1019-1024. doi: 10.1089/fpd.2009.0475