Oct 06, 2025

BONCAT_DECODE

This  protocol  is a draft, published without a DOI.
  • 1UKCEH;
  • 2UK Centre for Ecology and Hydrology
Susheel Bhanu Busi
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Protocol Citation: Tim Goodall, Susheel Bhanu Busi 2025. BONCAT_DECODE. protocols.io https://dx.doi.org/
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: February 14, 2024
Last Modified: October 06, 2025
Protocol  Integer ID: 95214
Keywords: decode analysis
Abstract
DECODE analysis
Protocol materials
Tween-20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P-7949
Sample prep and HPG incubation
17m
  1. Transfer 1 g of soil to a 50 ml falcon tube
  2. Add 5 ml 1X PBS and 5 sterile glass beads
  3. Vortex for 00:02:00
  4. Remove roots and add 20 ml 1X PBS
  5. Add 32 µL of 100 mg/ml HPG
  6. Incubate for 00:15:00 at room temperature
17m
Cell separation and fixation
1h
  1. Add 5 ml of 0.2% Tween-20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P-7949 in 1X PBS {see Probing_active_fraction_of_soil_microbiomes}
  2. Vortex for00:05:00
  3. Centrifuge at 500 g for 55 minutes {yes, you read it right - 00:55:00 }
  4. Collect supernatant into a fresh falcon tube
  5. Add 1:1 100% EtOH (final volume: 50 % (v/v) )
  6. Store at -20 °C for downstream processing

1h
Click reaction and staining
  1. Perform the `click reaction` as described in protocol by Moss et al.
  2. The collected cell population includes both active and inactive fractions (if sample in 50% ETOH dilute 2:3 in PBS)
  3. Staining with SYBR Green == "total population" (if sample in 50% ETOH dilute 1:49 in PBS prior to staining)
  4. HPG count == "active population"

Note
Run HPG count prior to SYBR green due to potential crossover events


Below are steps for field based HPG incorporation and preservation
Alternative approach for field base HPG incubation and cell fixation
1. Transfer up to 1g of soil/rhizosphere material to 50ml centrifuge tube
2. Add PBS at 25ml per gram material and 5 glass beads (e.g. if 0.4g add 10 mL )
3. Vortex 00:02:00
4. For rhizosphere samples remove roots by tipping slurry into new tube
5. Add 32 µL of 100 mg/ml HPG per gram material (e.g. if 0.4g add 12.8 µL )
6. Incubate for 00:15:00 at room temperature
7. Fix cells by 1:1 addition of 4% PFA (final concentration 2 % (v/v) )
8. Briefly mix and store refrigerated


17m
Cell separation, post field collection
1h
1. Add 0.2% Tween-20 in PBS at 5ml per gram material (e.g. if 0.4g add 2ml)
2. Vortex for00:05:00
3. Centrifuge at 500 g for 55 minutes {yes, you read it right - 00:55:00 }
4. Collect supernatant into a fresh falcon tube
1h
Click reaction
Refer to Moss et al