Immunoprecipitation (IP)

1. Solutions and Reagents
   Lysis buffer:
   1. Typically use RIPA buffer (25 mM Tris-HCl pH 7.6, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS). Proteinase inhibitor cocktail should be added fresh before each use.
   2. The usage of SDS depends on the nature of the cells. For some cell lines, 0.1% SDS will release DNA and thus make it hard to extract proteins out. In those cases, omit SDS
2. Preparation of cell lysates
   1. Add ice cold lysis buffer (1ml per 100mm-dish or 107 cells, or adjust based on your specific requirements). Scrape off cells (for adherent cells still on plate) and resuspend cells. Collect cells in a centrifuge tube and agitate for 30 min at 4°C.
   2. Spin cells at 4°C for 20 min at 12000 rpm.
   3. Save the supernatant which is the cell lysates.
3. Pre-clearing
   1. Add normal serum or irrelevant antibody from the same species and isotypes as the IP antibody you will use. The amount should be at least 5-fold more than the amount you will use for IP. Incubate for 1 hr at 4°C.
   2. For 1 ml lysate, add 100 ul of proteins A or protein G beads slurry (50 ul solid bed volume), and incubate at 4°C for 30 min on a rotator.
   3. Spin down beads at 14000g for 5 min at 4°C.
   4. Save the supernatant which is the pre-cleared lysates.
4. D. Immunoprecipitation (IP)
   1. Add IP antibody to the pre-cleared lysates. You will need to determine the best amount of antibody to use. As a starting point, you may use 1 ug antibody for every ml of lysates.
   2. Incubate for a certain amount of time (from 1 hr to overnight, depending on your specific conditions) at 4°C.
   3. Add 100 ul of protein A or protein G slurry (50 ul solid bed volume) to 1 ml lysate and incubate for 3 hr at 4°C on a rotator.
   4. Spin down beads, and remove supernatant.
   5. Wash beads 3 times with lysis buffer.
   6. Add SDS-PAGE sample buffer to beads. Boil and run gel.

FACS Protocols

Flow Cytometry for Intracellular Staining

1. Solutions and Reagents
   1. 1X Phosphate Buffered Saline (PBS): Dissolve 8g NaCl, 0.2g KCl, 1.15g Na2HPO4 and 0.2g KH2PO4 in 800mL distilled water (dH2O). Adjust the pH to 7.4 with HCl and the volume to 1 liter. Store at room temperature.
   2. Fixation buffer: 2% paraformaldehyde in 1xPBS
   3. Permeabilization buffer : 0.1% Triton X-100 in 1xPBS
   4. FACS buffer: 0.5% BSA , 0.05% Azide in 1xPBS
   5. Fluorescent dye conjugated secondary antibody.
2. Fixation
   1. Collect cells by centrifugation and aspirate supernatant.
   2. Fix the cell by 125μl cold fixation buffer, vortex briefly.
   3. Incubate at room temperature for at least 30 min or for 1hr 40C.
   4. Centrifuge for 5min at 300g,remove the supernatant.
3. Permeabilization
   1. Add 1ml permeabilization buffer to each tube.
   2. Centrifuge briefly, and aspirate supernatant.
   3. Resuspend cells in 125μl of permeabilization buffer and incubate at room temperature for 5min.
4. Staining
   1. Aliquot 1-2x106 cells into each tube.
   2. Add 1 ml FACS buffer to each tube, centrifuge to pellet the cells.
   3. Resuspend cell pellet with 125μl FACS buffer containing diluted primary antibody, vortex and incubate on ice for 30min.
   4. Rinse as before in FACS buffer by centrifugation.
   5. Resuspend cells in fluorescent dye conjugated secondary antibody, diluted in FACS buffer per manufacturer’s recommendations.
   6. Incubate for 30 minutes on ice.
   7. Rinse the cells as before in FACS Buffer by centrifugation.
   8. Resuspend cells in 0.5 ml PBS and analyze on flow cytometer

Immunofluorescent Staining Protocol

Immunocytochemistry

1. Solutions and Reagents
   1. 1X Phosphate Buffered Saline (PBS): Dissolve 8g NaCl, 0.2g KCl, 1.15g Na2HPO4 and 0.2g KH2PO4 in 800mL distilled water (dH2O). Adjust the pH to 7.4 with HCl and the volume to 1 liter. Store at room temperature
   2. Poly-L-lysine solution: 0.1mg/ml in 1xPBS
   3. Glass coverslips No.1, 18mm dia.
   4. Fixation buffer: 4% paraformaldehyde in 1xPBS
   5. Permeabilization buffer : 0.1% Triton X-100 in 1xPBS
   6. Blocking buffer: 5% Fetal Bovine Serum (FBS) in 1xPBS
   7. Fluorescence-labeled secondary antibody
2. Fixation permeabilization
   1. Coat the coverslips with 0.1mg/ml poly-L-lysine solution at room temperature for 2hrs, dry, and then wash with 1xPBS buffer.
   2. Place the coated coverslip into each well of 12-well plate, and inoculate cells the day before immunocytochemistry experiment.
   3. Suck off the medium and rinse cells attached to cover slips twice with 1xPBS, removing liquid by gentle aspiration in this and subsequent steps.
   4. Fix cells with 4% paraformaldehyde in 1xPBS for 6 min at room temperature, and then rinse briefly twice with 1xPBS.
   5. The fixed cells can be permeabilize with 0.1% Triton X-100 in 1xPBS for 6 min.
   6. Wash cells briefly twice with 1xPBS, then block the coverslip with blocking buffer briefly at R/T.
3. Staining
   1. Dilute primary antibody with blocking buffer, and incubate the coverslip for 60 min at room temperature.
      Note: You may wish to leave one slip for a secondary antibody only control.
   2. Wash cells 3 times with 1xPBS, then 2 times with blocking buffer.
   3. Incubate cells with a dilution of the fluorescence-labeled secondary antibody in blocking buffer for 30–45 minutes at room temperature in the dark.
   4. Wash cells three times with 1xPBS.
   5. Mount the coverslip on a glass slide. Store the slides in the dark.

IHC Protocols

Immunohistochemistry Protocol for Paraffin-embedded Tissues

1. Solutions and reagents
   1. Xylene
   2. Ethanol, anhydrous denatured, histological grade (100%, 95%, 70%)
   3. Washing buffer:
      TBST washing buffer: 1XTBS/0.1% Tween-20
      To prepare stock solution of 10X TBS: add 24.2 g Trizma base and 80 g sodium chloride to 1L of dH2O. Adjust pH to 7.6.
      Working solution: 1XTBST/0.1% Tween-20: add 100ml 10XTBS to 900 ml dH2O. Add 1 ml Tween-20 and mix well.
   4. Distilled water (dH2O)
   5. Antigen Retrieval Solution:
      0.01M Sodium Citrate Buffer, pH 6.0
      To prepare stock solutions: Solution A. 0.1 M citric acid solution: dissolve 21.0 g of citric acid, monohydrate (C6H8O7.H2O) in 100 ml of dH2O. Solution B. 0.1M sodium citrate solution: dissolve 29.4 g trisodium citrate dihydrate (C6H5Na3O7.2H2O) in 100 ml of dH2O.
      Working solution: Add 9 ml of Stock solution A and 41 ml of stock solution B to 450 ml of dH2O. Adjust pH to 6.0.
   6. 3% Hydrogene Peroxide
   7. Blocking buffer:
      1xPBS: This buffer is made by dissolving 8g of NaCl, 0.2g of KCl, 1.44g of Na2HPO4 and 0.24g of KH2PO4 into 800ml of distilled water. Then adjust the pH to 7.4 with HCl, and add H2O to 1 liter. Add 10% serum to make the final blocking buffer (serum origin depends on the host of the secondary antibody)
   8. Hematoxylin QS (catalog #H-3404 from Vector Laboratories, Inc.)
   9. Permanent Mounting medium (VectaMount, catalog# H-5000 Vector Laboratories, Inc.)
      
2. Protocol
   1. Deparaffinization/Rehydration
      1. Heat slides in an oven at 65 °C for 1 hour.
      2. De-paraffinize/hydrate using the following series of washes: two Xylene washes (5 min each), followed by two 100% ethanol rinses (5 min each), followed by 95% ethanol, 70% ethanol, 50% ethanol, 30% ethanol, followed by H2O and a TBST wash for 5 min on a shaker.
   2. Antigen Retrieval
      1. Immerse slides into staining dish containing Antigen Retrieval Solution.
      2. Place covered staining dish into the rice cooker. Add 120 mL d H2O and press “cook”.
      3. When “cook” is turned to “warm” (about 20–30 min), unplug the cooker and remove the staining dish to the bench top.
      4. Allow to cool down, without cover, for 20 min.
   3. Staining
      1. Wash slides with TBST for 5 min on a shaker.
      2. Inactivate endogenous peroxidase by covering tissue with 3% hydrogen peroxide for 10 min.
      3. Wash slides three times with TBST (3 min each on a shaker).
      4. Block slides with the blocking solution for 1 hour.
      5. Dilute primary antibody in the blocking buffer per recommendation on the data sheet.
      6. Apply primary antibody to each section and incubate overnight in the humidified chamber (4 ºC).
      7. Wash slides three times with TBST (3 min each on a shaker).
      8. Apply to each section secondary HRP-conjugated anti-rabbit antibody diluted in the blocking solution per manufacturer’s recommendation; incubate for 1 hour at room temperature.
      9. Wash slides three times with TBST (3 min each on a shaker).
      10. Add freshly prepared DAB substrate to the sections.
      11. Incubate tissue sections with the substrate at room temperature until suitable staining develops (generally 2–5 min).
      12. Rinse sections with water.
      13. Counterstain with Hematoxylin.
      14. Rinse sections with water.
      15. Dehydrate samples using two rinses with 100% Ethanol (20 dips per rinse) followed by two rinses with Xylene (30 dips per rinse).
      16. Mount coverslips on slides using Permount medium.

Western Blot Protocols

Western blot analysis

1. Run SDS-PAGE gel, and then Western transfer the protein samples to nitrocellulose (NC) membrane for immunoblot analysis.
2. After transfer, transfer the membrane to western-blot tray, briefly wash the NC membrane with distilled water.
3. (Optional) Visualize the proteins on the membrane by Ponceau’s staining.
4. Wash off the red stain with distilled water.
5. Block the membrane with 5-10ml blocking buffer (made by 5% non-fat milk in 1xPBST) for 30 minutes at R/T.
6. Dilute the primary antibody with blocking buffer according to the suggested dilution factor on datasheet (In case of anti-DDK mouse monoclonal antibody (TA100011, do 1:4000 dilution).
7. Remove the blocking buffer and add enough diluted primary antibody to cover the membrane.
8. Incubate the membrane with primary antibody for 1hr at R/T. (Note: Or you can do overnight incubation at 4C, make sure you cover the western-blot tray to prevent excessive evaporation). To prevent uneven coverage, the western-blot tray can be rocked on a rocker platform.
9. Collect the primary antibody and store them at 4C for up to two weeks. (If you would like to store them longer, you can freeze the diluted antibody at –20C. Remember frequent freezing and thawing will gradually decrease the antibody titer.)
10. Briefly wash the membrane with 1xPBST once to remove any excessive primary antibody.
11. Add enough 1xPBST to cover the membrane and leave the Western-blotting tray on a rocker platform.
12. Wash the membrane for 15 minutes. (Note: If the background is high, repeat this step for two to three times.), turn on the developer during the wash time.
13. Dilute HRP-conjugated secondary antibody with blocking buffer (1:5000 or higher dilution is usually good for Goat anti-mouse-HRP; TA100015).
14. Incubate the membrane with secondary antibody for 30 minutes to 1hr.
15. Wash the membrane with 1xPBST for 15 minutes, and then 3 times (5 min/time).
16. Prepare the chemiluminescence development substrate mixture by mixing equal amount of solution1 and 2 (TA100016; Normally 1ml will be enough for one membrane).
17. Prepare a plastic saran film, lay the film on a flat surface, and dispense 1ml of substrate mixture for one membrane on the plastic saran film.
18. Use a forceps to take washed the blot from the western-blotting tray, flip it, lay on the substrate mixture, and then incubate for 1 to 5 minutes. (Note: To avoid air bubbles, always lay the blot by touching one edge first.)
19. Remove excess Chemiluminescence Reagent and wrap the membrane in plastic. Place inside X-ray cassette.
20. Expose to film and develop

Buffer preparation

1xPBS: This buffer is made by dissolving 8g of NaCl, 0.2g of KCl, 1.44g of Na2HPO4 and 0.24g of KH2PO4 into 800ml of distilled water. Then adjust the pH to 7.4 with HCl, and add H2O to 1 liter.

1xPBST: 0.05% Tween 20 in 1xPBS

Reference

Sambrook, Fritsch, and Maniatis (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, volume 3, apendix B.12

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