Oct 07, 2021
HMA determination
- 12021 iDEC NEFU_China
- NEFU_China 2021
Protocol Citation: Shuning Guo 2021. HMA determination. protocols.io https://dx.doi.org/10.17504/protocols.io.byuupwww
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 07, 2021
Last Modified: October 07, 2021
Protocol Integer ID: 53876
Abstract
This protocol is used to determine the yield of HMA producted by E. coli by using HPLC.
Materials
HPLC (Agilent 1260)
Welch Xtimate C8 column (4.6 × 250 mm, 3.5 µm)
KH2PO4
100% acetonitril
ZY
50×M
50×5052
1000×Trace element
1M MgSO4·7H2O
antibiotic
20% arabinose
5 × M9
1 M CaCl2
20% glucose
ddH2O
0.22μm membrane filter
HPLC sample bottle
Centrifuge
Before start
Wash the sample bottle with ddH2O for 3 times and absolute ethanol for 1 time by using ultrasonic cleaner.
Prepare 8 mM KH2PO4 (pH 2.4) and filter it before use.
Activation of the cells
Activation of the cells
Inoculate 5 ml of LB medium with 1% volume of E. coli culture and culture at 37℃ for 12h.200 rpm, 37°C, 12:00:00
Note
Antibiotics of corresponding resistance should be added into the LB medium.
Induction of protein expression
Induction of protein expression
Prepare ZYM-5052 medium (5ml) by mix ingredients below: (for detailed recipe 
ZYM-5052.docx
ZYM-5052.docx | A | B | |
| ZY | 4800μl | |
| 50×M | 100μl | |
| 50×5052 | 100μl | |
| 1000×Trace element | 5μl/10μl | |
| 1M MgSO4·7H2O | 5μl/10μl | |
| antibiotic | 5μl | |
| 20% arabinose | 50μl |
Inoculate 5 ml of ZYM-5052 medium with 1% volume of E. coli LB culture and culture at 37℃ for 12h.200 rpm, 37°C, 12:00:00
Fermentation
Fermentation
Prepare M9 medium (10ml) by mix ingredients below:
| A | B | |
| 5 × M9 | 2ml | |
| 1M MgSO4·7H2O | 20μl | |
| 1 M CaCl2 | 1μl | |
| 20% glucose | 200μl | |
| ddH2O | 7.8ml |
Centrifuge 6OD bacteria in 1.5 ml eppendorf (EP) tube at 4,200 x rpm for 10 minutes at room temperature.
Decant or aspirate and discard the culture media.
Use 200μl M9 medium to spirate and dis completely resuspend cell pellet and culture at 37℃ for 12h.200 rpm, 37°C, 12:00:00
Sample preparation
Sample preparation
Centrifuge at 15,000 x g for 10 minute at room temperature.
Take 100 µl of supernatant into another EP tube and mix it with 900μl ddH2O.
Filter the sample with 0.22μm membrane filters and inject the sample into a clean sample bottle.
HPLC (high performance liquid chromatography)
HPLC (high performance liquid chromatography)
HMA was measured an HPLC (Agilent 1260) equipped with a UV detector and a Welch Xtimate C8 column (4.6 × 250 mm, 3.5 µm). The samples were analyzed under the following gradient of eluent A (8 mM KH2PO4, pH 2.4) and eluent B (100% acetonitril): 5 min 10% B, 20 min linear gradient to 30% B, 3 min linear gradient to 50% B, 5 min 50% B, 2 min linear gradient to 10% B, 5 min 10% B. HMA was detected at 215 nm.