Site-specific incorporation of BpF into ubiquitin-like proteins (UBLs) in E. coli

Zac Sandy; Zijuan Wang; Deepak Behera; Benjamin Foster; Finlay Martin; Kira Brüninghoff; Wolfgang Dörner; Kathleen Cain; Maria Jose Cabello-Lobato; Josep Forment; Matthew Cliff; Igor Larrosa; Perdita Barran; Duncan Smith; Henning Mootz; Christine Schmidt

Nov 22, 2024

Site-specific incorporation of BpF into ubiquitin-like proteins (UBLs) in E. coli

DOI

dx.doi.org/10.17504/protocols.io.5qpvo9yqbv4o/v1

Zac Sandy¹,
Zijuan Wang¹,
Deepak Behera¹,
Benjamin Foster¹,
Finlay Martin¹,
Kira Brüninghoff²,
Wolfgang Dörner²,
Kathleen Cain¹,
Maria Jose Cabello-Lobato¹,
Josep Forment³,
Matthew Cliff¹,
Igor Larrosa¹,
Perdita Barran¹,
Duncan Smith⁴,
Henning Mootz²,
Christine Schmidt¹

¹University of Manchester;
²Institute of Biochemistry, University of Muenster;
³Astra Zeneca, Cambridge;
⁴Cancer Research UK Manchester Institute

Benjamin Foster

University of Manchester

DOI: dx.doi.org/10.17504/protocols.io.5qpvo9yqbv4o/v1

Protocol Citation: Zac Sandy, Zijuan Wang, Deepak Behera, Benjamin Foster, Finlay Martin, Kira Brüninghoff, Wolfgang Dörner, Kathleen Cain, Maria Jose Cabello-Lobato, Josep Forment, Matthew Cliff, Igor Larrosa, Perdita Barran, Duncan Smith, Henning Mootz, Christine Schmidt 2024. Site-specific incorporation of BpF into ubiquitin-like proteins (UBLs) in E. coli. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo9yqbv4o/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: November 11, 2024

Last Modified: November 22, 2024

Protocol Integer ID: 112588

Keywords: Ubiquitin-like proteins (UBLs), Escherichia coli, BpF-incorporated protein

Funders Acknowledgement:

MRC research grant

Grant ID: MR/X008754/1

David Phillips Fellowship

Grant ID: BB/N019997/1

Deutsche Forschungsgemeinschaft

Grant ID: SFB858/B-14

Abstract

This protocol details the site-specific incorporation of BpF into ubiquitin-like proteins (UBLs) in E. coli. The use of a photo-crosslinkable residue within UBLs enhances the detection of weak and transient protein-protein interactions. This protocol provides a general workflow for the production of such protein probes, followed by methods to detect specific protein interactions using targeted or proteomic approaches. This protocol is linked to the PLOS ONE protocols manuscript "Site-selective photo-crosslinking for the characterisation of transient ubiquitin-like protein-protein interactions".

Materials

1 × SDS sample buffer 

AB
Tris-HCl pH 6.860 mM
SDS2%
glycerol10% 
bromophenol blue0.01% 
beta-mercaptoethanol1% 

Prescission protease buffer 

AB
Tris-HCl pH 7.550 mM
NaCl150 mM
EDTA1 mM
DTT1 mM

Protease mix 

AB
Tris-HCl pH 7.550 mM
NaCl150 mM
EDTA1 mM
DTT1 mM

Buffer A

AB
urea8 M
SDS2%
Tris100 mM
NaCl200 mM
pH 8adjusted with HCl

Buffer B 

AB
urea8 M
SDS0.2%
ethanol10%
isopropanol10%
Tris100 mM
NaCl1.2 M
pH 8adjusted with HCl

Buffer C 

AB
urea8 M
SDS0.2%
ethanol10%
isopropanol10%
Tris100 mM
NaCl200 mM
pH 5adjusted with HCl 
 
4.	Buffer D 

AB
urea8 M
SDS0.2%
ethanol10%
isopropanol10%
Tris100 mM
NaCl200 mM
pH 9adjusted with HCl
 

ReagentpEVOL-pBpFaddgeneCatalog #31190  
ReagentEZ-Link&trade; Maleimide-PEG2-BiotinThermo FisherCatalog #21901BID  
ReagentZeba&trade; Spin Desalting Columns, 7K MWCO, 0.5 mLThermo FisherCatalog #89883  
ReagentStreptavidin Sepharose High Performance, 5 mLCytivaCatalog #17511301  
 spin columns (Pierce Micro-Spin Columns, 10510824)
 ReagentTriethylammonium bicarbonate (TEAB)Merck MilliporeSigma (Sigma-Aldrich)Catalog #T7408  
Amount20 µL   PreScission Protease (Cytiva, #Reagent Prescission ProteaseGenscriptCatalog #Z02799 ), or suitable alternative

Transformation of E. coli with plasmids encoding the BpF-aminoacyl-tRNA synthetase/tRNA pair and the amber mutant of the UBL to express the BpF-incorporated protein

23h 43m

Mix Amount25 µL   of E. coli (BL21(DE3) or similar expression strain that does not have chloramphenicol resistance) chemically competent cells with Amount50 ng  –Amount100 ng   of pEVOL-pBpF (Addgene #31190) and 50–100 ng of an expression plasmid containing the protein of interest with the TAG amber codon for incorporating the unnatural photo-activatable amino acid 4-benzoyl-(L)-phenylalanine (BpF), in a microcentrifuge tube. Incubate TemperatureOn ice   for Duration00:30:00  .

Note
pEVOL-pBpF was a gift from Peter Schultz (Addgene plasmid #31190; RRID:Addgene_31190). Human ISG15 (uniprot ID: P05161) was expressed as the mature form (amino acids 2-157) with an additional C-terminal cysteine before the non-amber STOP codon to enable biotinylation. ISG15 cDNA was inserted into a pGEX-6P1 plasmid backbone using BamHI and XhoI restriction sites to have an N-terminal GST-3C tag. The C78S mutation and amber STOP (TAG) codons were prepared by site-directed mutagenesis. Plasmid maps and sequences are available upon request.

30m

Heat-shock the E. coli-plasmid DNA mixture by placing into a Temperature42 °C   water bath for Duration00:00:30   and then incubate TemperatureOn ice   for Duration00:01:00  -Duration00:02:00  .

Add Amount700 µL   LB broth or SOC media and incubate at Temperature37 °C   for Duration01:00:00   while shaking.

Plate Amount100 µL  -Amount200 µL   of the competent cells onto an LB agar plate supplemented with Amount50 undetermined   ampicillin and Amount34 undetermined   chloramphenicol. 

Incubate the LB agar plates at Temperature37 °C   DurationOvernight  .

The next day, grow a single transformed colony in Amount5 mL  -Amount25 mL   of LB broth supplemented with Amount50 undetermined   ampicillin and Amount34 undetermined   chloramphenicol at Temperature37 °C   DurationOvernight   with shaking.

Inoculate the overnight bacterial culture with ratio 1 in 100 into a 2 L flask and grow the E. coli culture for ∼Duration02:00:00   at Temperature37 °C   until the OD600 value reaches ∼0.6, as monitored by a spectrophotometer.

Note
Dissolve the unnatural amino acid photo-crosslinker (4-benzoyl-L-phenylalanine, BpF, CAS number: 104504-45-2) to Amount334 undetermined   (e.g. Amount0.27 g   in Amount3 mL   for a 500 ml LB volume) in Concentration1 Molarity (M)   NaOH solution while the culture is growing.

Once the OD600 value reaches ∼0.6, add 0.05% (w/v) arabinose, Concentration2 millimolar (mM)   BpF (v/v) and Concentration200 micromolar (µM)   IPTG as final concentration to induce the expression of the engineered UBL probe. Transfer the bacterial suspension to Temperature20 °C   for a further Duration12:00:00  -Duration16:00:00  .

Note
Expression conditions such as IPTG concentration, temperature, length of time can be optimised for the protein of interest.

18h

Harvest the bacteria by centrifuging at Centrifigation4.000 x g, 4°C, 00:10:00  and discard the supernatant. The pellet can be stored at Temperature-80 °C   or taken forward for lysis and downstream purification.

10m

Check protein expression

Centrifuge Amount1 mL   of E. coli culture grown from Go to   before and ~Amount250 µL   (approximately similar number of cells following overnight growth) of E. coli culture from Go to   after expression at Centrifigation13000 x g, Room temperature, 00:01:00 . 

Discard the supernatants and suspend the pellets with Amount100 µL   of 1 × SDS sample buffer. Heat the samples at Temperature95 °C   for Duration00:05:00  .

1 × SDS sample buffer 

AB
Tris-HCl pH 6.860 mM
SDS2%
glycerol10% 
bromophenol blue0.01% 
beta-mercaptoethanol1% 

Load Amount5 µL   of each sample onto a denaturing SDS-PAGE gel (homemade, percentage depends on predicted molecular weight (MW) of the protein of interest) and analyse by staining with Coomassie brilliant blue or Western blot if necessary.

Protein purification (GST affinity purification) and biotinylation

34m 30s

Note
Protein of interest purification can be achieved using any affinity or other approaches with additional downstream purification steps, if required. The example described here is for a GST-tagged protein expressed using the pGEX6P1 backbone plasmid followed by tag removal with 3C/Prescission protease.

Re-suspend the bacterial pellet in 5x pellet volumes of lysis buffer (e.g. if the pellet weighs 1 g, use Amount5 mL   lysis/binding buffer). Lysis buffer in this example is PBS Ph7.4  supplemented with Concentration1 millimolar (mM)   DTT and 1x protease inhibitors.

Sonicate the pellet TemperatureOn ice   at 40% amplitude (QSonica Q125 sonicator with a 3.2 mm diameter probe) for Duration00:04:00   total time with a Duration00:00:30   on, Duration00:00:30   off, cycle to lyse the cells.

Clarify the lysate by centrifugation at Centrifigation20000 x g, 4°C, 00:30:00  to pellet insoluble material and filter the supernatant through a 0.45 μm syringe filter to remove cellular debris.

30m

For a 1 mL GSTrap column (Cytiva), wash the column with 5 column volumes (CV) water using a 1 mL/min flow rate. Equilibrate the column with binding buffer by washing through 10 CV of cold buffer at a 1 mL/min flow rate.

Inject the lysate onto the column at a slow flow rate (~0.2-0.5 mL/min) and collect the flow-through.

Wash the column with Amount10 mL   (10 CV) of binding buffer at a flow rate of 1 mL/min.

Equilibrate the column with Amount10 mL   of Prescission protease buffer at a rate of 1 mL/min.

Prescission protease buffer 

AB
Tris-HCl pH 7.550 mM
NaCl150 mM
EDTA1 mM
DTT1 mM

Use a syringe adaptor to load Amount1 mL   of protease mix onto the column and incubate at Temperature4 °C   DurationOvernight   to remove the GST-tag.

Protease mix 

AB
Tris-HCl pH 7.550 mM
NaCl150 mM
EDTA1 mM
DTT1 mM

PreScission protease (Cytiva, #Z02799) or suitable alternative

30s

Collect the flow-through (containing untagged protein of interest) with Amount3 mL   of Prescission protease buffer.

Run samples of the lysate input and the eluted fractions on an SDS-PAGE gel to check for the yield and purity of the protein of interest.

Biotinylation of recombinant UBL probes

Biotinylation of the engineered cysteine was carried out using an EZ-Link biotin maleimide reaction kit (Sigma, #cat21901BID).

An overnight incubation was carried out according to the manufacturer’s instructions. Excess biotin was removed using Amount7 undetermined   MWCO Zeba spin desalting columns (ThermoScientific, #cat89883) equilibrated with PBS.

Another shorter Duration01:00:00   incubation was carried out according to the manufacturer’s instructions. Following the second incubation, excess biotin was again removed using 7 kDa MWCO Zeba spin desalting columns (ThermoScientific, #cat89883) equilibrated with PBS.

Successful biotinylation can be discerned by Western blot using streptavidin-HRP, affinity purifications using Streptavidin beads (see below), or by intact-mass spectrometry of the probe.

Photo-induced crosslinking with purified, recombinant UBL probes and binding partners

1h 20m

Incubate the BpF-containing UBL (a final concentration of Concentration20 micromolar (µM)   is a suitable starting point, followed by optimisation depending on the specific properties of the interaction between the UBL and its binding partner) with the respective binding partner (10 µM or 20 µM or optimised depending on affinity for the UBL) in PBS buffer for Duration00:15:00   at Temperature4 °C   in 0.2 mL thin-walled polypropylene PCR tubes, with a maximum volume of Amount50 µL  -Amount100 µL  .

15m

Divide the sample into two equal volumes, with half incubated at TemperatureRoom temperature  , and the other half irradiated for Duration01:00:00   with long-wave UV light (𝜆=365 nm; Herolab UV-16 L, 6 W, 6 mm distance).

Note
Photo-crosslinking time and temperature can be optimised depending on the protein of interest and target. The irradiated sample will be warmed by approximately Temperature3 °C   during incubation, but this does not lead to any visible precipitation in our experiments.

Following irradiation, add an equal volume of 2 × SDS sample buffer followed by heating for Duration00:05:00   at Temperature95 °C  . 
Check the crosslinked band by running an SDS-PAGE gel. 
The covalent bond remains under denaturing conditions and a band shift of the crosslinked proteins can be visualised by Coomassie-staining or Western blot.

Photo-induced crosslinking of UBL probes with proteins contained in cell extracts

1h 50m 20s

Whole cell extract can be prepared from any chosen or available cell types (e.g. HEK293T or HeLa cells). Cell lysate can be prepared using lysis buffer, with incubation TemperatureOn ice   for Duration00:20:00  , and centrifugation at Centrifigation17000 x g, 4°C, 00:10:00 . 
Alternatively, cells can be re-suspended in PBS supplemented with protease inhibitors and lysed by sonication (QSonica Q125 sonicator with a 2 mm diameter probe) TemperatureOn ice   for Duration00:00:20   at 30% amplitude 5 s on/5 s off, followed by centrifugation as above.

Lysis buffer 

AB
Tris-HCl pH 7.510 mM
NaCl150 mM
EDTA0.5 mM
MgCl22 mM
Nonidet P-400.5%
Benzonase2.5 units/µl
c0mplete EDTA-free protease inhibitors (Roche)1x

30m 20s

Incubate the biotinylated BpF-containing UBL (final concentration Concentration20 micromolar (µM)  ) and Amount2.5 undetermined  -Amount8 undetermined   (final concentration) whole cell extract, in a total volume of Amount100 µL   for Duration00:15:00   at Temperature4 °C   with rotation (e.g. roller or rotating wheel).

Note
The UBL proteins would be in PBS buffer pH 7.5, and the whole cell extract would be in lysis buffer (see above).

15m

Divide the sample into two parts. One part is incubated at TemperatureRoom temperature  , and the other part is irradiated for Duration01:00:00   with long-wave UV light (𝜆=365 nm; Herolab UV-16 L, 6 W, 6 mm distance).

After irradiation, add the same volume of 2 × SDS loading buffer to the samples followed by heating for Duration00:05:00   at Temperature95 °C  . The crosslinked band can be analysed by SDS-PAGE and Western blotting.

Enrichment of UBL binding partners from whole cell extract

4h 15m

Mix the biotinylated UBL probe (Concentration20 micromolar (µM)  ) with HEK293T or HeLa cell extract (Amount5 undetermined  -Amount10 undetermined  ) in PBS buffer supplemented with HALT protease inhibitor cocktail (Thermo Scientific) in a total volume of 100 µL and incubate for Duration00:15:00   at Temperature4 °C  .

15m

Divide the sample in half. Irradiate one part for Duration01:00:00   with long-wave UV light using a hand-held UV lamp (𝜆=365 nm; Herolab UV-16 L, 6 W, 6 mm distance). 

If necessary, perform this step at Temperature4 °C   to avoid overheating of the sample. Incubate the other part without UV irradiation for Duration01:00:00   at a comparable temperature.

Afterwards, add approximately Amount50 µL   streptavidin sepharose beads (Cytiva, 17-5113-01) added to each sample and incubate for Duration02:00:00   at Temperature4 °C   on a rotation wheel.

Note
Wash the beads beforehand 4 times with PBS buffer.

Wash the beads thoroughly 5 times with Amount500 µL   of each of the following buffers using spin columns (Pierce Micro-Spin Columns, 10510824):

1.	Buffer A

AB
urea8 M
SDS2%
Tris100 mM
NaCl200 mM
pH 8adjusted with HCl

2.	Buffer B 

AB
urea8 M
SDS0.2%
ethanol10%
isopropanol10%
Tris100 mM
NaCl1.2 M
pH 8adjusted with HCl

3.	Buffer C 

AB
urea8 M
SDS0.2%
ethanol10%
isopropanol10%
Tris100 mM
NaCl200 mM
pH 5adjusted with HCl 
 
4.	Buffer D 

AB
urea8 M
SDS0.2%
ethanol10%
isopropanol10%
Tris100 mM
NaCl200 mM
pH 9adjusted with HCl
 
Note
All urea buffers should be freshly prepared; buffer A: Duration00:02:00   incubation time on the beads before centrifugation; buffer B-D: Duration00:01:00   incubation time.

Wash the beads 10 times with Amount500 µL   buffer E (Amount50 undetermined   ammonium bicarbonate (NH4HCO3)) to ensure that the SDS is washed away properly and to prepare for on-bead or in-gel digestion (see below).

Note
Both on-bead and in-gel trypsin digestion were tested following streptavidin-mediated enrichment of UV-crosslinked proteins. Sample preparation choice depends on the protein of interest and available pipelines.

Mass spectrometry preparation and analysis

21h 9m

On-bead digestion

Transfer the streptavidin resins to a fresh reaction tube with a spatula and add Amount20 µL   of buffer E. 
For an on-bead tryptic digest, add DTT (Concentration5 millimolar (mM)   final concentration) [dissolved in freshly prepared Amount50 undetermined   NH4HCO3] and incubate for Duration00:30:00   at Temperature56 °C   while shaking.

30m

Add 2-iodoacetamide (IAA, Concentration25 millimolar (mM)   final concentration) [dissolved in freshly prepared Concentration50 millimolar (mM)   NH4HCO3] and incubate for Duration00:20:00   at TemperatureRoom temperature   in the dark.

20m

Add DTT (Concentration15 millimolar (mM)   final concentration) [quenching of IAA], and then add Amount200 ng   trypsin (e.g. Trypsin Gold, Promega or Sigma) dissolved in Amount10 µL   of Concentration50 millimolar (mM)   NH4HCO3.

After Duration00:10:00  , add Amount30 µL   of ProteaseMax (Promega) in Concentration50 millimolar (mM)   NH4HCO3 [0.1%] in water. Incubate the mixture DurationOvernight   at Temperature37 °C   on a rotation wheel.

20m

Acidify the supernatant with formic acid (FA), vacuum dry completely and resuspended in Amount7 µL   in acetonitrile/water (2% v/v), acidify with FA (0.1% v/v) and analyse by LC-MS/MS.

Note
Amount1 µL   for each technical repeat is used.

In-gel digestion

After washing, material was eluted by boiling in SDS sample buffer and loaded on a homemade 15% SDS-PAGE gel. 
Gel lanes were sliced into 5 mm horizontal slices and placed into separate 1.5 ml low bind Eppendorf tubes.

Gel slices were dehydrated with Amount900 µL   HPLC grade acetonitrile and incubation at TemperatureRoom temperature   for Duration00:15:00   with agitation (e.g. thermomixer at Centrifigation900 rpm ). 

The supernatant was removed before rehydration with 900 µl HPLC grade water and incubation at TemperatureRoom temperature   for Duration00:15:00   with agitation. 
This cycle of dehydration-rehydration was repeated a total of 5 times to ensure efficient removal of both aqueous and organic solvent soluble contaminants.

30m

The gel pieces were dehydrated a final time with Amount900 µL   HPLC grade acetonitrile and incubated as above. The supernatant was aspirated off and gel slices were dried to completeness in a vacuum centrifuge at Temperature45 °C  for Duration00:30:00  .

30m

Gel slices were rehydrated in Amount40 µL   Concentration50 millimolar (mM)   TEAB (#catT7408, Sigma) and sequencing grade trypsin (source as above) at a concentration of Amount20 undetermined  . 

Gel slices were incubated without shaking at TemperatureRoom temperature   for Duration00:20:00   before the addition of Amount100 µL   Concentration50 millimolar (mM)   TEAB and incubation at 37 °C with shaking (e.g. using a thermomixer) at Centrifigation900 rpm, 18:00:00 .

18h 20m

Overnight digests were acidified by the addition of formic acid to a final concentration of 0.2% (v/v). 

Digest supernatants from the same lane were pooled together and peptides dried to completeness in a vacuum centrifuge at Temperature45 °C  . 
Peptide digests were finally resuspended in Amount5 µL   0.2% formic acid immediately prior to LC-MS/MS analysis.

LC-MS/MS

LC-MS/MS was performed with use of a Vanquish Neo HPLC system coupled to an Orbitrap Lumos mass spectrometer via an EasySpray source (Thermo). The HPLC utilised mobile phases A (0.1% formic acid in water) and mobile phase B (80% acetonitrile, 0.1% formic acid).

Peptides were injected directly onto an Ionopticks reverse phase Aurora TS column (25 cm long, 75 µm ID) using a pressure/flow control protocol utilising a maximum pressure of 800 bar or 600 nl/min. 
Peptides were separated at 180 nl/min with a gradient of 1-4% B over Duration00:01:00   followed by 4-40% B% over Duration00:38:00  . 
LC eluent was sprayed directly into the MS source at an ion spray voltage of 1.7 Kv. 
The MS was operated in data independent acquisition (DIA) mode. 
An MS1 orbitrap scan was performed at 120K resolution with an m/z range of 350-1200 with a target value set to standard mode and injection time set to auto. 
Targeted orbitrap MS2 scans were set at 361.7, 384.1, 406.84, 428.8, 451.2, 473.5, 495.9, 518.3, 540.6, 563.0, 585.4, 607.7, 630.1, 652.5, 674.8, 697.2, 719.6, 719.9, 764.3, 786.7, 809.1, 831.4 and 853.8 using HCD normalised collision energy of 30% set to z 2 and a mass range of 23.5 Th. 
The Orbitrap MS2 scans were performed at a resolution of 15K (at m/z 200) with a normalised AGC target of 2000% and a max fill time of 22 ms. 
DIA data was processed using Spectronaut 17 (Biognosys) in direct DIA mode using oxidation (M), deamidation (NQ) and Acetylation (N term) as variable modifications in a search against a human Uniprot database.

39m

Acknowledgements

1 × SDS sample buffer 

AB
Tris-HCl pH 6.860 mM
SDS2%
glycerol10% 
bromophenol blue0.01% 
beta-mercaptoethanol1% 

Prescission protease buffer 

AB
Tris-HCl pH 7.550 mM
NaCl150 mM
EDTA1 mM
DTT1 mM

Protease mix 

AB
Tris-HCl pH 7.550 mM
NaCl150 mM
EDTA1 mM
DTT1 mM

Buffer A

AB
urea8 M
SDS2%
Tris100 mM
NaCl200 mM
pH 8adjusted with HCl

Buffer B 

AB
urea8 M
SDS0.2%
ethanol10%
isopropanol10%
Tris100 mM
NaCl1.2 M
pH 8adjusted with HCl

Buffer C 

AB
urea8 M
SDS0.2%
ethanol10%
isopropanol10%
Tris100 mM
NaCl200 mM
pH 5adjusted with HCl 
 
4.	Buffer D 

AB
urea8 M
SDS0.2%
ethanol10%
isopropanol10%
Tris100 mM
NaCl200 mM
pH 9adjusted with HCl
 

ReagentpEVOL-pBpFaddgeneCatalog #31190  
ReagentEZ-Link&trade; Maleimide-PEG2-BiotinThermo FisherCatalog #21901BID  
ReagentZeba&trade; Spin Desalting Columns, 7K MWCO, 0.5 mLThermo FisherCatalog #89883  
ReagentStreptavidin Sepharose High Performance, 5 mLCytivaCatalog #17511301  
 spin columns (Pierce Micro-Spin Columns, 10510824)
 ReagentTriethylammonium bicarbonate (TEAB)Merck MilliporeSigma (Sigma-Aldrich)Catalog #T7408  
Amount20 µL   PreScission Protease (Cytiva, #Reagent Prescission ProteaseGenscriptCatalog #Z02799 ), or suitable alternative

	A	B
	Tris-HCl pH 6.8	60 mM
	SDS	2%
	glycerol	10%
	bromophenol blue	0.01%
	beta-mercaptoethanol	1%

	A	B
	urea	8 M
	SDS	0.2%
	ethanol	10%
	isopropanol	10%
	Tris	100 mM
	NaCl	1.2 M
	pH 8	adjusted with HCl

	A	B
	Tris-HCl pH 7.5	10 mM
	NaCl	150 mM
	EDTA	0.5 mM
	MgCl2	2 mM
	Nonidet P-40	0.5%
	Benzonase	2.5 units/µl
	c0mplete EDTA-free protease inhibitors (Roche)	1x

Public workspaceSite-specific incorporation of BpF into ubiquitin-like proteins (UBLs) in E. coli

Site-specific incorporation of BpF into ubiquitin-like proteins (UBLs) in E. coli