Anti-Flag-tag Mouse 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.

Immunohistochemistry (Paraffin)

Deparaffinization/Rehydration

NOTE: Do not allow slides to dry at any time during this procedure.

1. Deparaffinize/hydrate sections:

1. Incubate sections in three washes of xylene for 5 min each.
2. Incubate sections in two washes of 100% ethanol for 10 min each.
3. Incubate sections in two washes of 95% ethanol for 10 min each.

2. Wash sections two times in dH2O for 5 min each.

Antigen retrieval

For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.

Staining

1. Wash sections in dH2O three times for 5 min each.
2. Incubate sections in 3% hydrogen peroxide for 10 min.
3. Wash sections in dH2O two times for 5 min each.
4. Wash sections in wash buffer for 5 min.
5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature.
6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C.
7. Remove antibody solution and wash sections with wash buffer three times for 5 min each.
8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature.
9. Wash sections three times with wash buffer for 5 min each.
10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use.
11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.
12. Immerse slides in dH2O.
13. If desired, counterstain sections with hematoxylin.
14. Wash sections in dH2O two times for 5 min each.
15. Dehydrate sections:
1. Incubate sections in 95% ethanol two times for 10 sec each.
2. Repeat in 100% ethanol, incubating sections two times for 10 sec each.
3. Repeat in xylene, incubating sections two times for 10 sec each.
16. Mount sections with coverslips and mounting medium.

Immunofluorescence (Immunocytochemistry)

Specimen Preparation (forcultured cell lines, IF-IC)

1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.
NOTE: Formaldehyde is toxic, use only in a fume hood.
2. Fix cells for 15 min at room temperature.
3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.
4. Proceed with Immunostaining.

Immunostaining

1. Add theblocking buffer and incubate for 60 min at RT.
2. Prepare primary antibody diluent in antibody dilution buffer as recommended .
3. Aspirate blocking solution, apply diluted primary antibody.
4. Incubate overnight at 4°C.
5. Rinse three times in 1X PBS for 5 min each.
6. Incubate specimens in fluorochrome-conjugated secondary antibody diluted in antibody dilution buffer for 1–2 hr at room temperature in the dark.
7. Rinse three times in 1X PBS for 5 min each.
8. Mount slides usingmounting medium with DAPI and cover with coverslips.
9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.

Flow Cytometry

Fixation

NOTE: lyse red blood cells and wash by centrifugation prior to fixation if using whole blood.
1. Collect cells by centrifugation and discardthe supernatant.
2. Resuspendthe cells in 0.5-1 ml 1X PBS and Add formaldehyde to obtain thefinal concentration of 4%.
3. Fix for 15 min at room temperature.
4. Wash by centrifugation with excess 1X PBS. Discard the supernatant and resuspendthe cells in 0.5-1 ml 1X PBS.

Permeabilization

1. Permeabilize the cells by adding ice-cold 100% methanol slowly, with gently vortexing.The final concentration of methanol is 90%.
2. Incubate the cells on the ice for 30 min.
3. Proceed with immunostaining or store the cells at -20°C in 90% methanol.

Immunostaining

NOTE: Count the cells using a hemocytometer or an alternative method.
1. Aliquot a desired number of cells into tubes or wells.
2. Wash the cellsin excess 1X PBS by centrifugation to remove methanol. Discard the supernatant. Repeat it if necessary.
3. Resuspendthe cells in 100 µl of diluted primary antibody prepared in incubation buffer.
4. Incubate for 1 hr at room temperature.
5. Wash the cells in incubation bufferand separated by centrifugation. Discard the supernatant. Repeat it twice.
6. Resuspendthe cells in 100 µl of diluted fluorochrome-conjugated secondary antibody.
7. Incubate for 30 min at room temperature.
8. Wash the cells in incubation buffer and removed the supernatant by centrifugation. Repeat it twice.
9. Resuspendthe cells in 1X PBS and analyze using flow cytometer (If the sample is also used for DNA staining, proceed to the next part).

E. Optional DNA Dye

1. Resuspendthe cells in DNA dye.
2. Incubate for at least 5 min at room temperature.
3. Analyze cells in DNA staining solution using flow cytometer.