PRODUCT-SPECIFIC PROTOCOLS

IP (DDX1)
Sample type:	SH-SY5Y cell
Amount of protein loaded:	30ug
Membrane:	NC
Transfer type:	Wet
Blocking buffer:	5% Non-fat Milk/ TBS
Primary antibody concentration:	0.5-2 μg/ml
Incubation time:	overnight at 4°C

PROTOCOL

1

Protein Extraction

A. Tissue Samples

Take 0.1–0.2 g tissue, mince finely.
Prepare RIPA lysis buffer and add protease/phosphatase inhibitors just before use.
Use 1 mL RIPA per 100 mg tissue.
Homogenize on ice using a tissue grinder.
Lyse at 4°C for 0.5–1 h, optionally apply sonication to enhance extraction.
Centrifuge at 10,000 rpm for 10 min, collect supernatant.

B. Cell Samples

Wash cells in 6-well plates with 200 μL pre-chilled PBS. Scrape cells and transfer to centrifuge tubes.
Prepare lysis buffer at RIPA:Protease Inhibitor:Phosphatase Inhibitor = 100:1:1.
Centrifuge at 3,000 rpm for 3 min, discard supernatant, add 5× volume lysis buffer, resuspend thoroughly. Lyse at 4°C for 0.5–1 h, sonicate if needed.
Centrifuge at 10,000 rpm for 10 min, collect supernatant.

2

Protein Quantification (BCA Method)

Prepare BCA working solution: mix 50 volumes BCA Reagent A with 1 volume Reagent B.
Microplate assay: Sample:BCA solution = 1:8 (or 1:20 if sample limited).
Incubate 37°C for 30 min, read absorbance at 562 nm.
Generate standard curve and calculate protein concentration according to dilution factor.

3

Protein Preparation

Adjust lysate to 3 mg/mL for Input group; remaining lysate to 1 mg/mL for IP experiments.

4

Protein A+G Magnetic Bead Preparation

Resuspend beads gently, transfer appropriate volume to a clean tube.
Wash beads 2–3 times with 1X TBS, resuspend to ~0.5 mL final volume.
Beads may clump; this is normal and does not affect experiment.

5

Antibody Binding to Beads

Prepare antibody working solution (5–50 μg/mL) in 1X TBS.
Optional: prepare control IgG solution for non-specific binding control.
Incubate beads with antibody or IgG at room temperature for 15 min – 1 h with rotation.
Wash beads 3× with 1X TBS, resuspend for subsequent IP.

6

Immunoprecipitation

Incubate lysate with antibody-bound beads (10–20 μL beads per 500 μL lysate) at room temperature for 2 h or 4°C overnight with rotation.
Separate beads on magnetic rack, keep supernatant if needed.
Wash beads 3× with 500 μL 1X TBS.
Optional: check OD280 of wash buffer to confirm removal of non-specific proteins.

7

Elution

Wash beads 3× with 1X TBST containing protease inhibitors.
Add 20 μL 5X SDS sample buffer with reducing agent, mix, and boil at 100°C for 5 min. Centrifuge at 10,000 g for 1 min, collect supernatant for SDS-PAGE or store at -20°C.

8

Western Blot Detection

Load 15–30 μL of sample on SDS-PAGE gel.
Separate proteins and transfer to NC membrane.
Detect target protein using suitable primary and secondary antibodies.