Western Blotting and RNA Isolation from Membrane
Eric L. Van Nostrand, Thai B. Nguyen, Chelsea Gelboin-Burkhart, Ruth Wang, Steven M. Blue, Gabriel A. Pratt, Ashley L. Louie, Gene W. Yeo
Abstract
Profiling of RNA binding protein targets in vivo provides critical insights into the mechanistic roles they play in regulating RNA processing. The enhanced crosslinking and immunoprecipitation (eCLIP) methodology provides a framework for robust, reproducible identification of transcriptome-wide protein-RNA interactions, with dramatically improved efficiency over previous methods. Here we provide a step-by-step description of the eCLIP method, along with insights into optimal performance of critical steps in the protocol. In particular, we describe improvements to the adaptor strategy that enables single-end enhanced CLIP (seCLIP), which removes the requirement for paired-end sequencing of eCLIP libraries. Further, we describe the observation of contaminating RNA present in standard nitrocellulose membrane suppliers, and present options with significantly reduced contamination for sensitive applications. These notes further refine the eCLIP methodology, simplifying robust RNA binding protein studies for all users.
Steps
Wash Beads (Prechill Buffers to 4°C)
Add 500µL, magnetically separate, remove the supernatant.
Add 500µL, mix, add 500µL, mix, remove the supernatant.
Wash 1× with 500µL, remove the supernatant.
Add 500µL, mix, add 500µL, mix, remove the supernatant.
Repeat wash 2× with . 500µL.
Prepare Samples for Gel Loading
Preparative gel —used for membrane transfer and RNA isolation.
Imaging gel —for IP-western blot imaging of IP success.
Remove supernatant, add 100µL, resuspend beads well.
Move 20µL to new tube #1 for Imaging gel IP samples .
Magnetically separate, remove the supernatant.
Resuspend sample in 20µL = Preparative gel IP samples .
Thaw On ice Preparative and Imaging input samples saved at Section "Remove Input Samples" under Capture RBP -RNA Complexes on Beads.
Prepare each sample mix:
| A | B |
|---|---|
| IP or input sample | 20 μL |
| 4× NuPAGE buffer | 7.5 μL |
| 1 M DTT | 3.0 μL |
Denature all samples in Thermomixer (1200rpm,70°C), and then cool On ice >0h 1m 0s.
For all samples , magnetically separate and only load supernatant (IP AND Inputs have beads).
Load and Run Gels
Load Preparative gel (4–12% Bis-Tris, 10-well, 1.5 mm) with (M) prestained markers and (m) diluted prestained marker (2µL + 2µL + 6µL) as follows for two experiments (A and B), leaving marker-only lanes between samples:
| A | B | C | D | E | F | G | H | I | J |
|---|---|---|---|---|---|---|---|---|---|
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| M | A-Input | (m) | A-IP | (m) | B-Input | (m) | B-IP | M | (m) |
- Load:
30µLfor all preparative samples (input and IP).
Load Imaging gel (4–12% Bis-Tris, 10 or 12-well, 1.5 mm).
- Load
15µL, save remaining volume at-20°Cas backup.
Run at 150 V in 1× MOPS running buffer for 1h 15m 0s or until dye front is at the bottom.
Transfer to Membranes
Prepare transfer: (use 4°C transfer buffer )
Imaging gel : Use PVDF membrane, activate with Methanol, and then move to transfer buffer .
Preparative gel : Use Nitrocellulose membrane ( see Note 4 4), presoak in transfer buffer .
Assemble transfer stacks, from bottom to top (black side (negative) of stack holder on bottom):
(negative)1× sponge – 2× Whatman – gel – membrane – 2× Whatman – 1× sponge (positive).
Transfer:
1h 15m 0s at 30 V (preferred) or 2 h at 200 mA.
After removing Preparative membrane, rinse quickly once with sterile 1× PBS, then wrap in Saran wrap and store at -20°C until Section "Cut Preparative Membrane (Cut Bands Based on Western Image)".
Develop Imaging Membrane
Block in 5% milk in TBST at Room temperature for 0h 30m 0s.
Probe with primary antibody at appropriate concentration (typically 0.2microgram per milliliter (μg/mL)) in 5% milk in TBST, at Room temperature for 1h 0m 0s.
Wash 3× with TBST, 0h 5m 0s:
Wash (1/3) with TBST, 0h 5m 0s.
Wash (2/3) with TBST, 0h 5m 0s.
Wash (3/3) with TBST, 0h 5m 0s.
Probe with secondary antibody: 1:4000 Rabbit TrueBlot HRP in 5% milk in TBST, incubate at Room temperature for 1h 0m 0s – 3h 0m 0s.
Wash 3× with TBST, 0h 5m 0s:
Wash (1/3) with TBST, 0h 5m 0s.
Wash (2/3) with TBST, 0h 5m 0s.
Wash (3/3) with TBST, 0h 5m 0s.
Develop with ECL, 0h 0m 30s to 0h 5m 0s, image with standard western blot film.
Cut Preparative Membrane (Cut Bands Based on Western Image)
Place preparative membrane on clean glass/metal surface.
Using a fresh razor blade, cut desired lane from the RBP band to 75 kDa above.
Slice membrane piece into ~1–2 mm slices.
Transfer slices to Eppendorf tube—place tube On ice if doing many samples.
Release RNA from Membrane
| A | B |
|---|---|
| ProK mix on ice, 200 μL per sample: | Urea/PK buffer |
| PK buffer: 160 μL | Dissolve 420 mg Urea in 500 μL PK buffer, then add PK buffer to a final volume of 1 mL |
| Proteinase K: 40 μL |
Add 200µL to membrane slices, incubate in Thermomixer (1200rpm,37°C).
Add 200µL to samples, mix, incubate in Thermomixer (1200rpm,37°C).
Purify RNA
Add 400µL (4.5), mix well, incubate in Thermomixer (1200rpm,37°C).
Transfer all except membrane slices to Phaselock gel (Heavy) tube, incubate in Thermomixer (1200rpm,37°C).
Centrifuge at 13000x g.
Transfer the aqueous (top) layer to a new 15 mL (or at least 3 mL volume) conical tube.
Add 2 volumes RNA binding buffer (typically 2× ~400 = 800µL).
Add equal volume 100% ethanol and mix (typically ~1200µL).
Transfer 750µL to Zymo-Spin column.
Centrifuge for 0h 0m 30s and discard flow-through.
Repeat spins by reloading additional 750µL volume until all sample has been spun through column.
Add 400µL, centrifuge for 0h 0m 30s, discard flow-through.
Add 700µL, centrifuge for 0h 0m 30s, discard flow-through.
Add 400µL, centrifuge for 0h 0m 30s, discard flow-through.
Centrifuge for additional 0h 2m 0s.
Transfer column to a new 1.5 mL tube (avoid getting wash buffer on column).
Add 10µL to column, let it sit for 0h 1m 0s, centrifuge for 0h 0m 30s.
Store CLIP samples at until Section "Reverse Transcribe RNA (All Clip and Input Samples), and Reaction Cleanup" (avoid multiple freeze-thaw cycles). -80°C until Section "Reverse Transcribe RNA (All Clip and Input Samples), and Reaction Cleanup" (avoid multiple freeze-thaw cycles).
