Project: Pou5f3 pulldown Page 1

2022.10.10 Hnrnpd Immunoprecipitation

Pou5f3 pulldown

Author: Edlyn Wu Created: 13.10.2022 13:32

Entry 34/34: 2022.10.10 Hnrnpd Immunoprecipitation Last modified: 18.10.2022 14:55
With tags: FLAG IP on Pou5f3:3xFLAG

To determine if Pou5f3 interacts with Hnrnpd when immunoprecipitate Hnrnpd.

Embryo collection
1. Prepare injection mix
1.32 μl hnrnpd:2xha RNA (prep date 14.05.2022) @ 378.12 ng/μl (100 ng/μl final)
0.25 μl Phenol Red
3.43 μl Nuclease-free water
5 μl total

2. Collect pou5f3:3xflag embryos after 15 mins mating.

3. Dechorionate embryos using Pronase.
4. Inject 1 puff (0.5 nL, 50 pg hnrnpd:2xha RNA)
5. Transfer embryos from injection plate to regular flat agarose plate.
6. Let embryos grow at 28°C until dome stage.
7. Once dome stage reached, transfer embryos in batches of 100 to low-binding tubes.
8. Buffer deyolk.
9. Remove all liquid, and flash-freeze cell pellets. Store at -80°C.
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HA Immunoprecipitation

All steps should be carried out on ice.

IP/lysis buffer 5 ml (- 10 ml 12 ml
det) (no PI) (+PI)

50 mM Tris-HCl pH 8 (1 M) 250 μl 500 μl 600 μl

150 mM NaCl (1 M) 750 μl 1.5 ml 1.8 ml

1 mM EDTA pH 8 (0.5 M) 10 μl 20 μl 24 μl

1% Triton X-100 (20%) - 500 μl 600 μl

1x Protease inhibitor (Roche, 25x) - 400 μl 480 μl

water 3990 6.05 ml 8.496

μl ml

Some notes about reagents:

For this experiment, I used some old reagents (past expiry date, but let's try)
HA antibody:
has still not arrived. Not enough to couple to beads for IP. Asked Sébastien (Gatfield lab), but they only have HA beads, but somehow they could not locate them.
Cell Signaling, C/N 3724S, L/N 8, 05/2016, clone C29F4
Protein G Dynabeads (Life Tech, C/N 10004D, L/N 167268080, expiry date: 2017.02 - inherited from Nouria Hernandez lab)

Embryo Pellet Preparation:

1. Thaw dome cell pellets on ice, then add 20 μl lysis buffer containing protease inhibitors to each tube. Let sit on ice for 20 mins.
IP date Background Collection date (# embryos) Concentration 5 mg for IP 400 ug for input Concentration of IP
pou5f3:3xflag 2022.10.11 (334), 2021.06.18 42.5 μ 95.2 μl 7.62 μl 5000μg / 500μl = 1mg
2020.04.23 (408) = 742 + 404.8 μl /ml for IP
buffer 20μl for UB and
(R) in 40μl for 5μ
pou5f3:3xflag + hnrnpd: 2022.10.10 (558), 2022.10.11 50 μ 100 μl 8 μl
2xha RNA (310) = 868 + 400 μl buffer

2. Homogenize with pre-chilled Dounce homogenizer (25 shots). Transfer homogenate to a 1.5 ml microtube, and homogenize with syringe 25G (10 strokes).

3. Spin at maximum speed at 4 °C 10 min

4. Transfer supernatant to a microtube and © at 4 °C, for 10 min. (repeat once more)

5. Transfer supernatant to a clean microtube and quantify by Qubit Protein assay.

6. Precipitate 200 μg of proteins with 3 volumes of acetone. Add 2x SDS-PAGE buffer to have a protein concentration of 5 μ. To be loaded on gel later… 400 μg / 5 μ = 80
μl

7. Protein bead equilibration:

Use 25 μl Dynabeads Protein G per sample (x2, 25 μl for pre-clear and another 25 μl for IP). To equilibrate, resuspend beads in 1 mL 1x TBS. Shake, place on magnet, and
discard supt. Repeat 3x. Then wash once with 1 mL lysis buffer. Shake, place on magnet, and discard supt. Resuspend beads in lysis buffer, same volume as the amount
removed from suspension.

8. Preclear: to 5 mg protein extract, add 25 μl Dynabeads in 500 μl lysis buffer. Let tubes rotate at 4°C for 1 hr. Place on magnet. Keep supt, discard beads.

9. Antibody binding: Add 10 μl HA antibody . Let tubes rotate at 4°C for 1 hr. Spin at max speed, 4°C for 5 mins.

10. IP: Transfer supt (extract-antibody mix) to new microtube. Add 25 μl beads. Let tubes rotate at 4°C for 1 hr.

11. Place tubes on magnetic rack and remove unbound fraction (Keep 200 μg for gel and precipitate with acetone). Wash beads-mix 4x in IP buffer (add same volume as
used in IP, vortex, and place on magnet). For the last wash, add lysis buffer (-det & protease inhibitors, same volume as used in IP) let rotate at 4 °C for 15 mins.

10. Elution: After last wash, aspirate supernatant carefully and add 20 μl 2x SDS-loading buffer to bead pellet.
Heat all samples for western blot analysis at 95°C for 5 mins, vortex, quick spin. Ready to load on gel.
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2022.10.13
Western blot analysis

Sample preparation
1. Heat samples at 95ºC, 5 mins
2. Vortex and quick spin

Gel electrophoresis and transfer

3. Load samples on 10% SDS-PAGE
Prestained Protein Plus Ladder: 3 μl
HA IP samples:
pou5f3:3xflag +hnrnpd:2xha RNA: 10 μl (out of 20 μl)
pou5f3:3xflag noninjected: 10 μl (out of 20 μl)
input samples:
pou5f3:3xflag +hnrnpd:2xha RNA: 10 μl (out of 80 μl)
pou5f3:3xflag noninjected: 10 μl (out of 80 μl)
unbound samples:
pou5f3:3xflag +hnrnpd:2xha RNA: 10 μl (out of 40 μl)
pou5f3:3xflag noninjected: 10 μl (out of 40 μl)
4. Migrate samples for 1 hr at 185 V.
5. Transfer at 25V, 1 hour onto nitrocellulose membrane.
6. Block with 5% milk in 0.1% PBS-T
7. 1ºAb O/N with: anti-FLAG: mouse, 1:1,000, F3165

2020.04.28
8. Wash 3x with 0.1% PBS-T
9. 2ºAb 45 mins with:
Fluorescent TrueBlot anti-mouse Ig DyLight 680 (Rockland Inc., C/N 18-4417-32)
10. Wash 3x with 0.1% PBS-T
11. Dry membrane for 1 hour
12. Licor

Try IP with ChIP protocol (nuclear fraction?)

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2022.10.14 hnrnpd_2xha_IP_compiled.png