Apr 25, 2025
Tandem affinity purification in Penicillium and Trichoderma
This protocol is a draft, published without a DOI.
- 1Shandong University
Protocol Citation: Yuqi Qin 2025. Tandem affinity purification in Penicillium and Trichoderma. protocols.io https://protocols.io/view/tandem-affinity-purification-in-penicillium-and-tr-dwkq7cvw
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: December 24, 2024
Last Modified: April 25, 2025
Protocol Integer ID: 117104
Abstract
Tandem affinity purification protocol for filamentous fungi Penicillium oxalicum and Trichoderma reesei
Tandem affinity purification in Penicillium and Trichoderma
Tandem affinity purification in Penicillium and Trichoderma
Prepare Cell Lysate
Fresh spore suspensions of the parent strain penicillium oxalicum or Trichoderma reesei with an HA-FLAG tag were inoculated into mycelium growth medium and incubated at 200 rpm, 30°C for 18-22 hours. The cultures were then transferred to 1L of media containing different carbon sources and further incubated at 200 rpm, 30°C on a shaker for 24 hours.The hyphae were filtered and washed by distilled water twice, then washed once with 0.96% NaCl (w/v) containing 1% DMSO and 1 mM PMSF, ground with liquid nitrogen, transferred to a 100 mL centrifuge tube, and added with 15 mL of protein lysis buffer (NaCl 9 g, 1M Tris-HCl, pH 7.5, glycerin 100 mL, and NP40 1 mL, per 1 L) and 0.05% protease inhibitor cocktail. After thoroughly mixing the mycelia and lysis buffer, the mycelia was placed on ice for 10 minutes. The samples were centrifuged at 9700 rpm and 4 °C for 30 min to obtain the suspension.
Bind to ANTI-FLAG resin
For each sample, 200 μL of IgG agarose beads were used, and 800 μL of WB250 was added to the beads. The mixture was centrifuged at 3000 g for 30 seconds at 4°C, followed by two washes. Subsequently, 800 μL of WB150 was added, and the beads were washed once.
The washed IgG agarose beads were transferred to a 50 mL centrifuge tube containing the protein solution and incubated with rotation at 4°C for 2–3 hours.
Near the end of the incubation, prepare 200 μL of ANTI-FLAG M2 affinity resin in a 1.5 mL centrifuge tube for each sample. Wash the resin according to the method used for Mouse IgG Agarose bead washing.
After sample incubation, centrifuge at 3000 rpm for 2 minutes at 4°C. Then, transfer the supernatant to a new 50 mL centrifuge tube. Subsequently, transfer the washed ANTI-FLAG M2 affinity resin into the 50 mL centrifuge tube containing the protein solution (in multiple small portions), and incubate with rotation overnight at 4°C.
Transfer and Wash Resin
Centrifuge at 3000 rpm for 2 minutes at 4°C, discard the supernatant, and transfer the beads completely into a 1.5 mL centrifuge tube (in multiple small portions).
Centrifuge at 3000 rpm for 30 seconds at 4°C and discard the supernatant. Then add 500 μL of cocktail and incubate with rotation at 4°C for 10 minutes. Centrifuge again at 3000 rpm for 30 seconds at 4°C and discard the supernatant. Repeat this process twice. Subsequently, transfer the beads completely into a centrifuge column (in multiple small portions), centrifuge at 3000 rpm for 30 seconds at 4°C, and discard the filtrate.
1st Elution of FLAG-HA-tagged Protein
Cover the bottom of the aforementioned centrifuge column with a red cap (avoid contamination during operation), add 250 μL of 3×FLAG peptide (final concentration 150 ng/μL), and incubate with rotation at 4°C for 10 minutes.
Remove the cap and collect the eluate in a new 2 mL centrifuge tube. Centrifuge at 3000 rpm for 1 minute.
Reattach the red cap to the bottom of the centrifuge column, add 250 μL of 3×FLAG peptide, and repeat the incubation for 10 minutes. Collect the eluate and mix it with the eluate from the previous step. Take 100 μL of the eluate for SDS-PAGE protein analysis, and use the remaining 400 μL of the eluate for the next step of anti-HA binding to the target protein.
Bind to Anti-HA resin
For each sample, prepare 40 μL of anti-HA resin in a 1.5 mL centrifuge tube. First, wash twice with 160 μL of WB250 by centrifuging at 3000 rpm for 30 seconds at 4°C. Then, wash once with 160 μL of WB150.
Transfer the anti-HA resin (in small multiple portions) into 400 μL of eluate, add 200 μL of cocktail, and incubate with rotation at 4°C for 2 hours.
Wash Resin
Centrifuge at 3000 rpm for 30 seconds at 4°C and discard the supernatant.
Add 500 μL of cocktail to the anti-HA resin beads and incubate with rotation at 4°C for 10 minutes.
Centrifuge at 3000 rpm for 30 seconds at 4°C and discard the supernatant. Repeat the incubation twice. Then transfer the beads to the centrifuge column, centrifuge at 3000 rpm for 30 seconds at 4°C, and discard the filtrate.
2nd Elution of Protein/protein Complex
Cover the bottom of the centrifuge column with a red cap, add 80 μL of 8 M urea, and incubate at room temperature for 10 minutes.
Remove the red cap, transfer the centrifuge column to a new 2 mL centrifuge tube, and centrifuge at 3000 rpm for 1 minute. Collect the eluate to obtain the second-step eluted protein solution.
Identify
Detect the presence of the tagged bait protein in the first-step and second-step eluates using Western blot, with either anti-HA or anti-FLAG as an antibody.
The other part of the eluate was assayed through LC-MS/MS to determine the putative interacting proteins from the bait protein.