TITLE: Western Blotting and RNA Isolation from Membrane
AUTHORS: Eric L. Van Nostrand, Thai B. Nguyen, Chelsea Gelboin-Burkhart, Ruth Wang, Steven M. Blue, Gabriel A. Pratt, Ashley L. Louie, Gene W. Yeo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div></div>

[STEPS]
?. [Wash Beads (Prechill Buffers to 4°C)]
Add , magnetically separate, remove the supernatant.
[Wash buffer]
?. [Wash Beads (Prechill Buffers to 4°C)]
Add , mix, add , mix, remove the supernatant.
[Wash buffer]
[High salt wash buffer]
?. [Wash Beads (Prechill Buffers to 4°C)]
Wash 1× with , remove the supernatant.
[High salt wash buffer]
?. [Wash Beads (Prechill Buffers to 4°C)]
Add  , mix, add , mix, remove the supernatant.
[High salt wash buffer]
[Wash buffer]
?. [Wash Beads (Prechill Buffers to 4°C)]
Repeat wash 2× with .
[Wash buffer]
?. [Prepare Samples for Gel Loading]
Preparative gel—used for membrane transfer and RNA isolation.
?. [Prepare Samples for Gel Loading]
Imaging gel—for IP-western blot imaging of IP success.
?. [Prepare Samples for Gel Loading]
Remove supernatant, add , resuspend beads well.
[cold Wash buffer]
?. [Prepare Samples for Gel Loading]
Move  to new tube #1 for Imaging gel IP samples.
[(20% of sample)]
?. [Prepare Samples for Gel Loading]
Magnetically separate, remove the supernatant.
?. [Prepare Samples for Gel Loading]
Resuspend sample in  = Preparative gel IP samples.
[ cold Wash buffer]
?. [Prepare Samples for Gel Loading]
Thaw  Preparative and Imaging input samples saved at Section "Remove Input Samples" under Capture RBP -RNA Complexes on Beads.
on ice
?. [Prepare Samples for Gel Loading]
Prepare each sample mix:  AB1IP or input sample20 μL24× NuPAGE buffer7.5 μL31 M DTT3.0 μL
AB1IP or input sample20 μL24× NuPAGE buffer7.5 μL31 M DTT3.0 μL
?. [Prepare Samples for Gel Loading]
Denature all samples in Thermomixer (), and then cool  >.
Centrifuge: 1200 33, 10 min, 70 10
on ice
?. [Prepare Samples for Gel Loading]
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 ( +  + ) as follows for two experiments (A and B), leaving marker-only lanes between samples:  ABCDEFGHIJ1123456789102MA-Input(m)A-IP(m)B-Input(m)B-IPM(m)Load:  for all preparative samples (input and IP).
[marker]
[4× NuPAGE buffer]
[Wash Buffer]
ABCDEFGHIJ1123456789102MA-Input(m)A-IP(m)B-Input(m)B-IPM(m)
[(all) volume]
?. [Load and Run Gels]
Load Imaging gel (4–12% Bis-Tris, 10 or 12-well, 1.5 mm).Load , save remaining volume at  as backup.
[all cold samples]
-20 °C
?. [Load and Run Gels]
Run at 150 V in 1× MOPS running buffer for  or until dye front is at the bottom.
?. [Transfer to Membranes]
Prepare transfer: (use  transfer buffer)
4 °C
?. [Transfer to Membranes]
Imaging gel: Use PVDF membrane, activate with Methanol, and then move to transfer buffer.
?. [Transfer to Membranes]
Preparative gel: Use Nitrocellulose membrane (see Note 4), presoak in transfer buffer.
?. [Transfer to Membranes]
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 to Membranes]
Transfer:
?. [Transfer to Membranes]
at 30 V (preferred) or 2 h at 200 mA.
?. [Transfer to Membranes]
After removing Preparative membrane, rinse quickly once with sterile 1× PBS, then wrap in Saran wrap and store at  until Section "Cut Preparative Membrane (Cut Bands Based on Western Image)".
-20 °C
?. [Develop Imaging Membrane]
Block in 5% milk in TBST at  for .
0 Room temperature
?. [Develop Imaging Membrane]
Probe with primary antibody at appropriate concentration (typically ) in 5% milk in TBST, at  for .
0 Room temperature
?. [Develop Imaging Membrane]
Wash 3× with TBST, :
?. [Develop Imaging Membrane]
Wash (1/3) with TBST, .
?. [Develop Imaging Membrane]
Wash (2/3) with TBST, .
?. [Develop Imaging Membrane]
Wash (3/3) with TBST, .
?. [Develop Imaging Membrane]
Probe with secondary antibody: 1:4000 Rabbit TrueBlot HRP in 5% milk in TBST, incubate at  for  – .
0 Room temperature
?. [Develop Imaging Membrane]
Develop with ECL,  to , image with standard western blot film.
?. [Develop Imaging Membrane]
Wash 3× with TBST, :
?. [Develop Imaging Membrane]
Wash (1/3) with TBST, .
?. [Develop Imaging Membrane]
Wash (2/3) with TBST, .
?. [Develop Imaging Membrane]
Wash (3/3) with TBST, .
?. [Cut Preparative Membrane (Cut Bands Based on Western Image)]
Place preparative membrane on clean glass/metal surface.
?. [Cut Preparative Membrane (Cut Bands Based on Western Image)]
Using a fresh razor blade, cut desired lane from the RBP band to 75 kDa above.
?. [Cut Preparative Membrane (Cut Bands Based on Western Image)]
Slice membrane piece into ~1–2 mm slices.
?. [Cut Preparative Membrane (Cut Bands Based on Western Image)]
Transfer slices to Eppendorf tube—place tube  if doing many samples.
on ice
?. [Release RNA from Membrane]
AB1ProK mix on ice, 200 μL per sample:Urea/PK buffer2PK buffer: 160 μLDissolve 420 mg Urea in 500 μL PK buffer, then add PK buffer to a final volume of 1 mL3Proteinase K: 40 μL
AB1ProK mix on ice, 200 μL per sample:Urea/PK buffer2PK buffer: 160 μLDissolve 420 mg Urea in 500 μL PK buffer, then add PK buffer to a final volume of 1 mL3Proteinase K: 40 μL
?. [Release RNA from Membrane]
Add  to membrane slices, incubate in Thermomixer ().
[ProK mix]
Centrifuge: 1200 33, 20 min, 37 10
?. [Release RNA from Membrane]
Add  to samples, mix, incubate in Thermomixer ().
[Urea/PK buffer]
Centrifuge: 1200 33, 20 min, 37 10
?. [Purify RNA]
Add  (), mix well, incubate in Thermomixer ().
[acid phenol/chloroform/isoamyl alcohol]
Centrifuge: 1200 33, 5 min, 37 10
?. [Purify RNA]
Transfer all except membrane slices to Phaselock gel (Heavy) tube, incubate in Thermomixer ().
Centrifuge: 1200 33, 5 min, 37 10
?. [Purify RNA]
Centrifuge at .
Centrifuge: 13000 34, 15 min
?. [Purify RNA]
Transfer the aqueous (top) layer to a new 15 mL (or at least 3 mL volume) conical tube.
?. [Purify RNA]
Add 2 volumes RNA binding buffer (typically 2× ~400 = ).
800 µl
?. [Purify RNA]
Add equal volume 100% ethanol and mix (typically ~).
1200 µl
?. [Purify RNA]
Transfer  to Zymo-Spin column.
[mixed sample]
?. [Purify RNA]
Centrifuge for  and discard flow-through.
?. [Purify RNA]
Repeat spins by reloading additional  volume until all sample has been spun through column.
750 µl
?. [Purify RNA]
Add , centrifuge for , discard flow-through.
[RNA Prep Buffer]
?. [Purify RNA]
Add , centrifuge for , discard flow-through.
[RNA Wash Buffer]
?. [Purify RNA]
Add , centrifuge for , discard flow-through.
[RNA Wash Buffer]
?. [Purify RNA]
Centrifuge for additional .
?. [Purify RNA]
Transfer column to a new 1.5 mL tube (avoid getting wash buffer on column).
?. [Purify RNA]
Add  to column, let it sit for , centrifuge for .
[H2O]
?. [Purify RNA]
Store CLIP samples at  until Section "Reverse Transcribe RNA (All Clip and Input Samples), and Reaction Cleanup" (avoid multiple freeze-thaw cycles).
-80 °C