xCT Rabbit Recombinant mAb

Western Blotting

Sample preparation

1. Aspirate media from cultures and Wash the cells with 1X PBS.
2. Lyse cells by adding 1X SDS sample buffer and transfer the extract to a microcentrifuge tube. Keep onice.
3. Sonicate for 10–15 sec to complete cell lysis and shear DNA.
4. Heat a 20 µl sample to 95–100°C for 5 min, then cool on ice.
5. Centrifuge for 5 min (with Microcentrifuge).
6. Load appropriate volumes of samples onto SDS-PAGE gel (loading quantity of protein sample depends on the concentration of extracted proteins).
NOTE: At the same time, please load the pre-stained molecular weight markers to determine molecular weights and verify electrotransfer.
7. Electrotransfer to nitrocellulose/PVDF membrane.

Membrane Blocking and Antibody Incubations

a. Blocking

1. (Optional) After transfer, wash the transferred membrane with TBS for 5 min at room temperature.
2. Incubate the membrane in the blocking buffer for 1 hr at room temperature.
3. Wash three times for 5 min each with TBST.

b. Antibodies Incubation

1. Incubate membrane and primary antibody (at the appropriate dilution and diluent recommended) in a primary antibody dilution buffer with gentle agitation overnight at 4°C.
2. Wash three times for 5 min each with TBST.
3. Incubate membrane with an appropriate second antibodydissolved in the blocking buffer with gentle agitation for 1 hr at room temperature.
4. Wash three times for 5 min each with TBST.
5. Proceed with detection.

Detection of Proteins

1. After antibodies incubation, Wash membrane three times for 5 minutes in TBST.
2. PrepareECL Reagent (or other chromogenic agents/substrate according to your second antibody). Mix well.
3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.

IP

Sample preparation

1. To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold 1X PBS.
2. Remove PBS and add ice-cold 1X cell lysis buffer to each plate and incubate on the ice for 5 min.
3. Scrape cells off the plate and transfer to themicrocentrifuge tubes. Keep on the ice.
4. Sonicate on the ice three times for 5 sec each.
5. Centrifuge for 10 min at 4°C, 14,000 x g and transfer the supernatant to a new tube. If necessary, store itin -80°C.

Cell Lysate Pre-Clearing (Optional)

Pre-clearing the lysate can help reduce non-specific binding and reduce background.

1. Vortex to mix beads.
2. Add 10–30 µl of 50% Protein Aagarose bead slurry to 200 µl cell lysate at 1 mg/ml.
3. Incubate with rotation at 4°C for 30–60 min.
4. Centrifuge for 10 min at 4°C. Transfer the supernatant to a fresh tube.
5. Proceed to the next part.

Immunoprecipitation

1. Add primary antibody at the appropriate dilutionto cell lysat. Incubate with gentle agitation or rotation overnight at 4°C.
2. Add protein Aagarose(10–30 µl of 50% bead slurry). Incubate with gentle agitation or rotation for 1–3 hr at 4°C.
3. Centrifuge for 30 sec at 4°C and discard the supernatant. Wash the pellet five times with 500 µl of 1X cell lysis buffer. Keep on the ice.
4. Continue with sample analysis.

Sample Analysis

a. Western Immunoblotting

1. Resuspend the pellet with SDS sample buffer. Vortex, then centrifuge for 30 sec at 14,000 x g.
2. Heat the samples to 95–100°C for 2-5 min and centrifuge for 1 min at 14,000 x g.
3. Load the appropriate amount ofsamples on a 4–20% gel for SDS-PAGE.
4. Analyze sample by western blot.

b. Kinase Assay

1. Wash the pellet twice with 500 µl 1X kinase buffer. Keep on the ice.
2. Suspend pellet in 40 µl 1X kinase buffer supplemented with 200 µM ATP and appropriate substrate.
3. Incubate for 30 min at 30°C.
4. Terminate the reaction by adding SDS sample buffer. Vortex to mix, then centrifuge for 30 sec.
5. Transfer supernatantcontaining phosphorylated substrate to another tube.
6. Heat the sample to 95–100°C for 2–5 min and centrifuge for 1 min at 14,000 x g.Load the sample on SDS-PAGE.