May 18, 2026

Sortase A-mediated farnesylation of a protein in vitro

DOI
https://dx.doi.org/10.17504/protocols.io.dm6gp1575gzp/v1
  • Sophie Tschirpke1,
  • Nynke M. Hettema1,
  • Liedewij Laan1
  • 1TU Delft
Sophie Tschirpke
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DOI: https://dx.doi.org/10.17504/protocols.io.dm6gp1575gzp/v1
Protocol Citation: Sophie Tschirpke, Nynke M. Hettema, Liedewij Laan 2026. Sortase A-mediated farnesylation of a protein in vitro. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp1575gzp/v1
Manuscript citation:
Tschirpke, S*.; Hettema, N. M.*; Spitzbarth, B; De Geus, M. A. R.; Van Opstal, F.; Eelkema, R.; Laan, L: Sortase A-mediated farnesylation of Cdc42 in vitro; ACS Synthetic Biology 2026, doi.org/10.1021/acssynbio.6c00136.

Copyright 2026 The Authors. Published by American Chemical Society. This publication is licensed under CC-BY-NC-ND 4.0.
*equally contributing authors
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 11, 2026
Last Modified: May 18, 2026
Protocol  Integer ID: 243037
Keywords: farnesylation, prenylation, protein modification, Sortase A, Sortagging, mediated farnesylation, step ligate triclycine cysteamine farnsyl, accessible farnesylation, farnesylation, reaction for the farnesylation, tagged protein, sortase recognition motif, farnesylated pois, purification tag, terminus of the protein, expressed protein, enzyme, protein, affinity chromatography, glycine in the motif, protein of interest, variable amino acid, labeling reaction, sortase, nucleic acid, formation of an amide bond, threonine of the substrate, amide bond, bond between threonine
Funders Acknowledgements:
European Research Council (Horizon 2020)
Grant ID: 758132
Nederlandse Organisatie voor Wetenschappelijk Onderzoek
Grant ID: 016.Vidi.171.060
Kavli Institute of Nanoscience Delft
Grant ID: Kavli Synergy Post-doctoral Fellowship
Abstract
This protocol describes a Sortase A-based approach to farnesylate a protein of interest (POI) in vitro. This method leverages E. coli-expressed protein with a Sortase A recognition motif, facilitating efficient and accessible farnesylation and purification using a purification tag-based strategy.

In brief: E. coli expression system are used to produce a double-tagged protein construct with the Sortase recognition motif flanked with two purification tags (C-terminal His-tag and another other N-terminal tag) [Tschirpke et al., 2023]. In the labeling reaction, commercially available Sortase A enzyme is used to cleave the C-terminal His-tag off the POI and in one step ligate triclycine cysteamine farnsyl [Tschirpke et al., 2026] to the C-terminus of the protein. The reaction mixture is cleaned by size exclusion chromatography (SEC), optionally followed by His-affinity chromatography (His-AC), to isolate the farnesylated POI.

Background: Transpeptidase Sortase A, derived from S. aureus, can label proteins in a precise site-specific manner due to its ability to perform both cleavage and ligation in one step. Sortase A recognizes a LPXTG motif (where L is leucine, P is proline, X is a variable amino acid, T is threonine, and G is glycine) in a substrate, cleaves the bond between threonine and glycine in the motif, and subsequently catalyzes the formation of an amide bond between the threonine of the substrate and the N-terminal glycine of a compatible probe [Antos et al., 2017; Guimaraes et al., 2013]. Sortase A has almost no requirements for the structure or sequence of the probe as long as it contains oligoglycine at the N-terminus. Sortase A has been used for many different labeling purposes ranging from labeling with fluorescent dyes, nucleic acids, or lipid chains as well as protein-protein and protein-membrane conjugations both in vivo and in vitro [Pritz et al., 2007; Antos et al., 2008; Sakamoto et al., 2010; Chen et al., 2016; Vilen et al., 2023]. In this protocol we utilize a Sortase A-mediated reaction for the farnesylation of a POI in vitro.
Attachments
Icon for Sortase_reaction_calculator.xlsx
Sortase_reaction_cal...
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Materials
  • Protein of interest (POI): POI needs to contain a needs to contain a C-terminal Sortase recognition motif followed by a His-tag, and an N-terminal purification tag (that is not a His-tag). Example protein sequences and their construction are given in [Tschirpke et al. 2023]. The steps in this protocol are shown for a POI with an N-terminal Flag-tag and a C-terminal His-tag.
  • Triglycine cysteamine farnesyl (might be commercially available, or can be synthesized [Tschirpke et al., 2026])
  • Sortase A, His-tagged (e.g. Sortase A Octamutant, BPS Bioscience)
  • Materials for SDS-PAGE protein gels and western blot analysis (I. anti-His, II. anti-POI or anti-Flag)
  • For clean-up steps 1 & 2: Chromatography columns and operating equipment for size-exclusion chromatography (SEC) and His-affinity chromatography (His-AC)
  • Low-binding tubes and pipette tips (e.g. Protein LoBind Tubes (Eppendorf), Low Binding TripleA pipette tips (Westburg))
  • Buffer components: Tris-HCl, NaCl, MgCl2, CaCl2, imidazole, 2-mercaptoethanol, ultrapure water
  • DMSO
  • 10% Brij58 (Surfact-Amps 58, Thermo Scientific)
  • Dithiothreitol (DTT)
  • Glycerol
Protocol materials
Dithiothreitol (DTT)
DMSO
Sortase A OctamutantBPS Bioscience
10% Brij58 (Surfact-Amps 58)Thermo Scientific
Troubleshooting
Problem
No reaction product (Flag-POI-farnesyl)
Solution
(1) Make sure the POI has an accessible C-terminal Sortase A recognition motif. Test the reaction with other Sortase probes. (2) Ensure that detergents (such as Brij58) were added to the reaction mixture and to the buffers to increase the solubility for farnesylated POI. If needed, try other detergents (see [Kai et al. 2023]). (3) Use low-binding plastics, the reaction product may be lost due to non-specific binding. (4) Remove GTP/ATP from the reaction buffer, as this can lead to precipitation of the product. (5) Add calcium to the reaction buffer, as many Sortase A variants require calcium. (6) Reduce the DMSO content, as a too high DMSO can reduce Sortase A activity.
Considerations beforehand
(Reaction: In the reaction, Sortase A enzyme cleaves the C-terminal purification-tag off the POI and in one step ligates triclycine cysteamine farnsyl to the C-terminus of the protein. The steps in this protocol are shown for a protein of interest (POI) with both an N-terminal Flag-tag and a C-terminal His-tag.
  • educt: Flag-POI-His
  • desired product: Flag-POI-farnesyl
  • undesired side-product: Flag-POI
  • reaction-catalyzing enzyme: His-Sortase

Educts and products of the Sortase A-mediated farnesylation reaction.
(Reprinted (adapted) with permission from [Tschirpke et al. 2026].)

Note
The protocol can also be used for differently tagged POIs. However, the presence/absence of purification tags can influence the analysis method (western blotting) and clean-up strategy used (see below).

Analysis method:

Small observable size shift between reaction educt and product on SDS-PAGE: During the reaction, Sortase A cleaves the C-terminal His-tag (0.8 kDa) off the POI, and ligates triglycine cysteamine farnesyl (0.45 kDa) to the POI. The expected size shift combined with a changed mobility due to increased hydrophobicity in the farnesylated POI can lead to a small distinctive shift on SDS-PAGE, which can provide a rough preliminary estimate for successful farnesylation. However, this should always be followed up with more clear-cut analysis methods (described below).

Anti-farnesyl western blot analysis can be challenging: Ideally, product formation can be detected using anti-farnesyl western blot analysis. We explored this analysis strategy, but could not make it work, every antibody combination we used lead to both false-positive and false-negative results. We suggest starting and further optimizing the protocols described in [Kennedy et al., 2019; Li et al., 2020].

Utilization of the purification tags for western blot analysis: To detect the desired product (Flag-POI-farnesyl), we utilized a combination of western blots: (1) anti-His and (2) anti-POI (alternatively, anti-Flag can also be used):
  1. anti-His western blot: signals which bands on SDS-PAGE contain the educt (Flag-POI-His) and Sortase A.
  2. anti-POI western blot (or anti-Flag western blot): signals which bands on SDS-PAGE contain one of the POI species: educt, undesired side-product, desired product.

Combination of the small size-shift on SDS-PAGE and both western blots allows for the identification of each reaction component:
  • Sortase A: visible both on anti-His blot
  • educt (Flag-POI-His): visible both on anti-His and anti-POI blot
  • undesired side-product (Flag-POI): visible on anti-POI blot
  • desired product (Flag-POI-farnesyl): visible on anti-POI blot

Both the desired and undesired products are only visible on the anti-POI western blot, where they appear as two separate bands. To identify which band corresponds to which product, a control reaction without triglycine cysteamine farnesyl is used. This control produces only the undesired side-product (Flag-POI).
By comparing the western blot of the farnesylation reaction to that of this control reaction, the band of the undesired side-product can be identified (it's the band that is visible in the control reaction). The other band corresponds to the desired product.


An example of how western blot analysis in combination with the control reaction can be used to identify the reaction components on SDS-PAGE. The POI here is Cdc42. (Reprinted (adapted) with permission from [Tschirpke et al. 2026].)
Key observations:
- The educt (Flag-Cdc42-His) is visible both in anti-His and anti-Cdc42 blot (diamond).
- The control reaction leads to one reaction product (Flag-Cdc42, filled diamond), visible only in anti-Cdc42 blot. it's the lower of the 2 bands in the 72h sample.
- The desired product (Flag-Cdc42-farnesyl, red dot) only gives a signal in the anti-Cdc42 blot.
- The presence of Flag-Cdc42-farnesyl is only very obvious in the SEC sample (only a signal in the anti-Cdc42 blot), after Flag-Cdc42-His is removed.
- Flag-Cdc42-farnesyl runs at almost the same height as Flag-Cdc42-His, therefore its presence is not very obvious until Flag-Cdc42-His is absent.

Intact protein mass analysis: The purification tag-based western blot analysis only gives indirect proof of the presence of farnesylated POI. We additionally used intact protein mass analysis to confirm the formation of the farnesylation product. We advise to use a mass-analysis facility that has expertise in detecting lipidated proteins, as otherwise the detection can easily fail due to farnesyl's 'sticky' properties.
We tried several in-house mass analysis methods, all of which failed, until sending our sample to a suitable facility (for details, see [Tschirpke et al. 2026]).

Clean-up strategy: This protocol utilizes size-exclusion chromatography (SEC), with an optional second clean-up step using His-affinity chromatography (His-AC).

Example of a clean-up protocol: During SEC, Flag-POI-farnesyl elutes in peak 1. Peak 2 contains all 3 POI species (Flag-POI-farnesyl, Flag-POI, Flag-POI-His) and Sortase A. SEC Peak 2 is further cleaned by His-AC, where educt (Flag-POI-His) and Sortase enzyme are removed. (Figure adapted from [Tschirpke et al. 2024].)
Clean-up step 1 utilizes SEC, during which reaction educt and products and the Sortase A enzyme are separated. In our experience the first SEC peak contains only the desired product (Flag-POI-farnesyl). The second peak contains all 3 POI species: desired product (Flag-POI-farnesyl), the undesired side-product (Flag-POI), and the remaining educt (Flag-POI-His). The third SEC peak contains leftover triclycine cysteamine farnsyl ('farnsyl'). Depending on the size difference of POI and Sortase A enzyme, Sortase A can be present in peak 1, peak 2, or elute as a separate peak.
If needed, clean-up step 2 (His-AC) can be used to further remove all His-tagged proteins (Flag-POI-His, and Sortase A enzyme). This step is advised if Sortase A is present in any of the peaks containing Flag-POI-farnesyl, as Sortase A can also catalyze the backwards reaction of removing the farnesyl group from the product. During His-AC, His-tagged proteins bind to the nickel beads, while proteins without a His-tag remain in the flow-throught (Flag-POI-farnesyl, Flag-POI).

Note
Utilization of His-tags for clean-up step 2 (His-AC): This protocol utilized that only the reaction educt (Flag-POI-His) and Sortase enzyme are His-tagged, allowing for their removal through His-AC.
If
  • Sortase A and POI exhibit a significant size difference, allowing for their separation using SEC, and
  • the mayority of educt is farnesylated during the reaction (almost no educt remains),
then clean-up step 2 (His-AC) is not necessary.

This means that
  • a Sortase A variant with or without a His-tag can be used
  • the choice of purification tags on the POI is unrestrained: E.g. a POI without any His-tag can be used, or the POI can contain a N-terminal His-tag. The use of a double-tagged POI remains advantageous for analysis (see ).

Preparation
To follow this protocol, ensure to have these materials and components on-hand:
Materials:
  • Low-binding tubes and pipette tips: e.g. Protein LoBind Tubes (Eppendorf), Low Binding TripleA pipette tips (Westburg)
Note
The farnesyl group easily sticks to plastic tubes. The use of 'normal' plastic tubes and pipette tips will lead to a loss in yield do to farnesyl educts and farnesylated product sticking to plastic. The use of low-binding tubes and pipette tips reduces this loss, increasing the reaction's yield.
  • For analysis: Materials for SDS-PAGE protein gels and western blot analysis (I. anti-His, II. anti-POI or anti-Flag)
  • For clean-up steps 1 & 2: Chromatography columns and operating equipment for size-exclusion chromatography (SEC) and His-affinity chromatography (His-AC)
Samples & chemicals:
  • purified POI : POI needs to contain a needs to contain a C-terminal Sortase recognition motif followed by a His-tag, and an N-terminal purification tag (that is not a His-tag). Example protein sequences and their construction are given in [Tschirpke et al. 2023]. The steps in this protocol are shown for a POI with an N-terminal Flag-tag and a C-terminal His-tag.
Note
The protocol can also be used for differently tagged POIs. However, the presence/absence of purification tags influences the analysis method (western blotting, ) and clean-up strategy used (see ).
This protocol utilized that only the reaction educt (Flag-POI-His) and Sortase enzyme are His-tagged, allowing for their removal through His-AC (optional clean-up step 2).

  • triglycine cysteamine farnesyl (might be commercially available, or can be synthesized [Tschirpke et al., 2026])
  • Sortase A, His-tagged (e.g. Sortase A OctamutantBPS Bioscience )
  • DMSO
  • 10% Brij58 (Surfact-Amps 58)Thermo Scientific
  • Dithiothreitol (DTT)
Prepare Buffers:
  • SEC buffer: 50 mM Tris-HCl (pH=7.5), 100 mM NaCl, 10 mM MgCl2, 1 mM 2-mercaptoethanol.
  • SEC buffer supplemented with 0.1% Brij58: 50 mM Tris-HCl (pH=7.5), 100 mM NaCl, 10 mM MgCl2, 1 mM 2-mercaptoethanol, 0.1% Brij58.
  • CaCl2 in SEC buffer: 100 mM CaCl2, 50 mM Tris-HCl (pH=7.5), 100 mM NaCl, 10 mM MgCl2, 1 mM 2-mercaptoethanol.
  • His-AC washing buffer: 50mM Tris-HCl (pH=8.0), 1 M NaCl, 5 mM imidazole, 1 mM 2-mercaptoethanol.
  • His-AC elution buffer: 50 mM Tris-HCl (pH=8.0), 100 mM NaCl, 500 mM imidazole, 1 mM 2-mercaptoethanol.

Note
  • For the labeling reaction, 'SEC buffer' and 'CaCl2 in SEC buffer' are needed.
  • Clean-up step 1 (SEC) uses the same buffers as the labeling reaction and 'SEC buffer supplemented with 0.1% Brij58'.
  • For clean-up step 2 (His-AC), 'His-AC washing buffer' and 'His-AC elution buffer' need to be prepared. These buffers can also be supplemented with 0.1% Brij58 if a lot of farnesylated product is lost during this step.

Dialyze the purified POI into SEC buffer.

Labeling reaction
Dissolve the reaction components:
Dissolve triglycine cysteamine farnesyl in DMSO at a final concentration of 20 mM.
E.g. Dissolve 5 mg triglycine cysteamine farnesyl in 500 uL DMSO.
Note
Prepare a fresh solution for each reaction, do not store dissolved triglycine cysteamine farnesyl. In our experience it might also degrade when stored in solution at -80°C.

Prepare fresh solution of 1 M Dithiothreitol (DTT) .
E.g. Dissolve 7.7 mg DTT in 500 uL ultrapure water.
Prepare the reaction mixture:
Mix Sortase A OctamutantBPS Bioscience , purified POI , and triglycine cysteamine farnesyl (dissolved in DMSO, ) at a 2 : 100 : 10’000 molar ratio, with a final DMSO concentration of 22%. Supplement the reaction with a final conc. of 20 mM CaCl2, 1% Brij58 and 2 mM freshly prepared Dithiothreitol (DTT) ( ).

Note
Recommended reaction supplements:
  • DTT - a reducing agent.
  • Brij58 - a detergent, added to solubilize the farnsylated POI. For reference and for comparison of Brij58's performance with other detergents, see [Kai et al. 2023].
  • CaCl2 - most Sortase A enzymes require the presence of calcium ions for their activity. If the presence of calcium ions is problematic, Sortase A mutants that function even without calcium ions can be used [Chen et al., 2016].

Discouraged reaction supplements:
  • ATP/GTP: In labeling reactions of ATPases/GTPases (as POIs) often ATP/ GTP is added. The hydrolysis of ATP/GTP by the POI during the labeling reaction releases free phosphate, which reacts with calcium ions to calcium phosphate, which has a low solubility and precipitates. This precipitation process can also precipitate POI out of solution, reducing the yield. If addition of ATP/GTP is absolutely required, CaCl2 should not be added and a Sortase A mutant that functions without calcium ions should be used (see above).

Note
Ratio of reaction components: In our experience, a molar ratio of 2 : 100 : 10’000 of Sortase A : POI : triglycine cysteamine farnesyl works well, but these values still have significant potential for optimization.
To do so, prepare small volumes of reaction mixture with varied molar ratios and take samples at various time points during the reaction. Monitor product formation / educt reduction through SDS-PAGE and western blot analysis.

Monitoring the reaction progress can be challenging:
  • The small observable size shift between reaction educt and product on SDS-PAGE might be insufficient to monitor product formation: see
  • Anti-farnesyl western blot analysis can be challenging: see
  • Reactions using other triglycine probes only provide approximations: To optimize the ratios, we replaced the triglycine cysteamine farnesyl with a fluorescently labelled pentaglycine peptide (Alexa Fluor488 C5 Maleimide, Invitrogen) ligated to Gly-Gly-Gly-Gly-Gly-Cys peptide (Biomatik)). We monitored product formation using the fluorescent signal of Alexa488 [Tschirpke, 2022]. When utilizing this strategy, we suggest to use a glycine probe with similar properties as the triglycine cysteamine farnesyl, ideally a Gly-Gly-Gly-Cys ligated to a hydrophobic and fluorescent chain. The best proxy for triglycine cysteamine farnesyl would be a fluorescently tagged triglycine cysteamine farnesyl (which we have not tried yet). To explore this strategy, we suggest reading [Kai et al. 2023].

DMSO content: With increasing concentrations of triglycine cysteamine farnesyl, the overall DMSO content also rises, which may affect Sortase A activity. Sortase A has been reported to retain full activity in the presence of up to 20% (v/v) DMSO, while a significant decrease in activity was observed at 40% solvent concentration [Pritz et al., 2007]. Because different Sortase A mutants exhibit varying intrinsic activities, their sensitivity to DMSO may also differ. Therefore, if high triglycine cysteamine farnesyl concentrations (and consequently high DMSO concentrations) are required, the activity of the specific Sortase A mutant used should be evaluated for the relevant DMSO concentrations.

Use the spreadsheet Sortase_reaction_calculator.xlsx to calculate the pipetting scheme for the reaction mixture.

Note
Reaction volume: If the reaction mixture should be cleaned by SEC ( ), choose a total reaction volume that is close to the maximum sample volume of the SEC column.

Note
Example reaction:
Pipetting scheme for an example reaction.
Steps:
Enter the concentrations of existing stocks and buffers:
  • For the Sortase A stock an exemplary conc. of 320 uM is entered into the table - this conc. is variable and depends on your starting solution (yellow cell).
  • For the POI an exemplary conc. of 94 uM is entered into the table - this conc. is variable and depends on your starting solution (yellow cell).
  • Triglycine cysteamine farnesyl was dissolved in DMSO at a conc. of 20 mM (see ).
  • CaCl2 in SEC buffer has a CaCl2 conc. of 100 mM (see ).
  • DTT was dissolved in ultrapure water at a conc. of 1 M (see ).
  • 10% Brij58 (Surfact-Amps 58)Thermo Scientific has a stock-conc. of 10%.

Enter the volume of POI used for this reaction (orange cell). The final volume is 2,25 times the POI volume. For this example, 250 uL were entered.

Enter the reaction component ratios (green cells). For this reaction, we use a molar ratio of Sortase A : POI : triglycine cysteamine farnesyl of 2 : 100 : 10'000.

Enter the final concentrations of the reaction supplements (dark blue cells): The reaction mixture contains 20mM CaCl2, 2 mM DTT, and 1% Brij58.

Enter the final DMSO concentration (light blue cell): 22%

Based on these values, the spreadsheet calculates the pipetting scheme:
Mix 1.5 uL Sortase A, 250 uL POI, 117.5 uL triglycine cysteamine farnesyl (in DMSO). Add 112.5 uL CaCl2 in SEC buffer, 1.1 uL 1M DTT, 56.3 uL 10% Brij58, 6.2 uL DMSO, and 17.4 uL SEC buffer.


Mix the reaction components based on the calculations of the previous step. Take a sample for SDS-PAGE analysis immediately after all components were added ('0h sample': 30 uL reaction sample + 10uL 4x SDS loading buffer/ Laemmli buffer [Laemmli, 1970]).

Note
Order of mixing: It is recommended to add triglycine cysteamine farnesyl (in DMSO), DMSO, and Sortase A as the last components.
  • DMSO is an organic solvent, which may (or may not) affect the POI's functionality when added in high concentrations. Adding DMSO-containing solutions at the end ensures that they diluted to the final concentration immediately after their addition.
  • Sortase A catalyzes the ligation reaction. In the first step, it cleaves the C-terminal His-tag from the POI. If a glycine-containing probe is present, Sortase A resolves this intermediate by ligating the probe to the POI. In the absence of a glycine probe, the reaction is prematurely terminated, resulting in an undesired reaction product. Thus, the presence of Sortase A without triglycine cysteamine farnesyl may lead to accumulation of this unwanted reaction product. Although this effect is unlikely to be significant in most cases, it can be readily avoided by adding Sortase A as a last component to the reaction mixture.

Incubate the reaction mixture for 72 h at 4◦C under constant slight agitation. At the end of the reaction, take a sample for SDS-PAGE analysis ('72h sample': 30 uL reaction sample + 10uL 4x SDS loading buffer/ Laemmli buffer [Laemmli, 1970]).

Directly continue to clean-up step 1 (SEC) or flash-freeze the samples for storage (after addition of 10% glycerol).

Note
Incubation duration and temperature:
We found that 72h at 4°C lead to optimal product formation - only ~5% of the educt remained [Tschirpke et al. 2026].
However, similar to the optimization of reaction component ratios (see ), the incubation conditions (time, temperature) can be further optimized. Initially, we had used a reaction with a fluorescently labelled pentaglycine peptide to explore different reaction conditions. Here we used the fluorescent signal to monitor product formation [Tschirpke, 2022].

Analyze the reaction using SDS-PAGE and western blotting (anti-His, anti-POI or anti-Flag).
  • The 0h sample should contain only a band of the educt (Flag-POI-His) and Sortase A (if visible at all, given the low concentration in the reaction mixture).
  • The 72h sample should contain a band for the desired product (Flag-POI-farnesyl) and a band of the undesired product (Flag-POI). A band for the educt may be visible, depending if all educt reacted or not. A band for Sortase A may be visible too.

Use the combination of anti-His and anti-POI western blotting to identify which bands correspond to which reaction component (see ).
It is key to distinguish the desired product (Flag-POI-farnesyl) from the undesired side-product (Flag-POI). Both are only visible on the anti-POI western blot, where they appear as two separate bands. To identify which band corresponds to which product, run a control reaction without triglycine cysteamine farnesyl. This control produces only the undesired side-product (Flag-POI). By comparing the western blot of the farnesylation reaction with that of this control reaction, the band of the undesired side-product can be identified (it's the band that is visible in the control reaction). The other band corresponds to the desired product.

Expected result
An example analysis using Cdc42 as POI is shown in .

Clean-up step 1: Size-exclusion chromatography (SEC)
Use SEC to separate the reaction components.
In our experience, this step is sufficient to isolate the desired reaction product (Flag-POI-farnsyl).
Set up the equipment and equilibrate the column in SEC buffer supplemented with 0.1% Brij58 ( ).
Ideally, have the column already ready when the reaction is finished, so the sample can be directly loaded onto the column.
Load the sample onto the column and elute using SEC buffer supplemented with 0.1% Brij58, collect the elution peaks. The desired product (Flag-POI-farnesyl) can be part of of one or two SEC peaks (see ).
Directly continue to clean-up step 2 (His-AC) or flash-freeze the samples for storage (after addition of 10% glycerol).

Note
Effect of detergents on SEC:
We added 1% Brij58 to the reaction mixture and used SEC buffer supplemented with 0.1% Brij58. We also tried
  • a reaction & SEC buffer without detergent
  • adding 5% CHAPS to the reaction mixture & using SEC buffer supplemented with 0.5% CHAPS

Both lead to less farnesylated product. Most noticeably, instead of observing 2 SEC peaks (as with the addition of Brij58), only 1 SEC peak was visible. Its position resided with that of SEC peak 2 (Brij58 condition).
In presence of Brij58 SEC peak 1 contained only farnesylated POI and SEC peak 2 contained all 3 POI species (Flag-POI-His, Flag-POI, Flag-POI-farnesyl). The presence of only SEC peak 2 in conditions without detergent/ with CHAPS suggests that most of farnesylated product was lost/ did not stay soluble (consistent with literature [Kai et al. 2023]).
[Kai et al. 2023] also discusses the effectiveness of other detergents for solubilizing farnesylated and geranyl-geranylated proteins.

Effect of the presence of detergents on SEC. (Figure adapted from [Tschirpke et al. 2024].)


Analyze the samples using SDS-PAGE and western blotting (see ).

Expected result
In our experience SEC results in 3 elution peaks; the first SEC peak contains only the desired product (Flag-POI-farnesyl). The second peak contains all 3 POI species - desired product (Flag-POI-farnesyl), the undesired side-product (Flag-POI), the remaining educt (Flag-POI-His) - and Sortase A. The third SEC peak contains leftover triclycine cysteamine farnsyl ('farnsyl') and cleaved His-tags.
Below the elution profile and SDS-PAGE and western blotting analysis of a farnesylation reaction using Cdc42 as POI are shown.

Example of a SEC elution profile. (Figure adapted from [Tschirpke et al. 2024].)


SDS-PAGE and western blot analysis of farnesylation reactions with Cdc42 as POI. (Reprinted (adapted) with permission from [Tschirpke et al. 2026].)
Key observations:
- The educt (Flag-Cdc42-His) is visible both in anti-His and anti-Cdc42 blot (diamond).
- The undesired side-product (Flag-Cdc42, filled diamond), is visible only in anti-Cdc42 blot and is the lower of the 2 bands in the 72h sample (confirmed by a control reaction).
- The desired product (Flag-Cdc42-farnesyl, red dot) only gives a signal in the anti-Cdc42 blot
- The presence of Flag-Cdc42-farnesyl is only very obvious in the SEC peak 1 sample (only a signal in the anti-Cdc42 blot), after Flag-Cdc42-His is removed.
- Flag-Cdc42-farnesyl runs at almost the same height as Flag-Cdc42-His, therefore its presence is not very obvious until Flag-Cdc42-His is absent.
- SEC peak 2 contains all 3 Cdc42 species. The blots alone are not proof to show that Flag-Cdc42-farnesyl is present in SEC peak2, confirmation can be obtained by (1) intact protein mass-spectrometry analysis, (2) use His-AC to remove remaining Flag-Cdc42-His.


[optional] Clean-up step 2: His-affinity chromatography (His-AC)
If needed, His-AC can be used to remove educt (Flag-POI-His) and His-tagged Sortase A from the sample. Load the sample onto a Nickel column (where the educt (Flag-POI-His) and His-tagged Sortase A will bind to the column material) and collect the flow-through.

Example of using His-AC as a second clean-up step after SEC. (Figure adapted from [Tschirpke et al. 2024].)

Set up the equipment and equilibrate the column in SEC buffer ( ).
Load the sample and collect the flow-through (which contains the cleaned-up sample).

After addition of 10% glycerol, the samples can be flash-frozen for storage.

Note
If not all His-tagged reaction components are removed in this step, use a column with a larger sample volume, reduce the loading speed, or load the sample repeatedly.
Note, loading the sample repeatedly can decrease the reaction yield, as it gives Flag-POI-farnesyl more chances to non-specifically bind to plastic surfaces (see ) .


[optional] Elute the proteins bound to the column using His-AC elution buffer ( ).
This step is only needed to check the effectiveness of His-AC (i.e. to collect samples for SDS-PAGE and western blot analysis), as Flag-POI-farnesyl is in the flow-through.
Analyze the samples using SDS-PAGE and western blotting.

Expected result
The signal of bands for His-tagged Sortase A and educt (Flag-POI-His) decreases in the flow-through samples. Ideally, these 2 components are removed from the flow-through sample.

SDS-PAGE and western blot analysis of farnesylation reactions with Cdc42 as POI. Here His-AC was performed for SEC peak 2. (Figure adapted from [Tschirpke et al. 2024].)
Key observations:
- The educt (Flag-Cdc42-His) is visible both in anti-His and anti-Cdc42 blot (diamond).
- The undesired side-product (Flag-Cdc42, filled diamond), is visible only in anti-Cdc42 blot and is the lower of the 2 bands in the 72h sample (confirmed by a control reaction).
- The desired product (Flag-Cdc42-farnesyl, red dot) only gives a signal in the anti-Cdc42 blot.
- The presence of Flag-Cdc42-farnesyl is only very obvious in the His-AC sample (only a signal in the anti-Cdc42 blot), after Flag-Cdc42-His is removed.
- Flag-Cdc42-farnesyl runs at almost the same height as Flag-Cdc42-His, therefore its presence is not very obvious until Flag-Cdc42-His is absent.



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Acknowledgements
We thank B. Spitzbarth, M.A.R. de Geus, and R. Eelkema for synthesizing triglycine cysteamine farnesyl (see [Tschirpke et al., 2026]). We thank R. van der Valk and F. van Opstal for experimental assistance and and the group of A. Jakobi (TU Delft) for their help in the exploration of prenylation methods. We thank N. Dekker (TU Delft) for the plasmid pET28a-His-mcm10-Sortase-Flag. Intact protein mass analysis was performed by the Mass Spectrometry Facility at Max Perutz Labs using the VBCF instrument pool.