StrippingWesternBlots

1) After ECL development, wash membrane once for 10min with PBST.

2) Incubate the membrane in stripping buffer (see below) in a heat-sealed plastic bag for 30min at 50oC with occasional mixing. For low-abundance antigens, the strip can be done overnight at RT with constant agitation.

3) Washed stripped membrane at least 3x for 10min each with PBST, then re-block (in PBST+NFDM or PBST+BSA) overnight at 4oC.

Stripping Buffer:            62.5mM Tris pH 6.8

                                    2% SDS

                                    100mM b-ME

Quick Mix:       Use 4X stacking buffer at 1:8, 10% SDS at 1:5, and b-ME at 70μl/10ml

                        (e.g.10mls strip=1.25mls 4X, 2mls 10% SDS, 70μl b-ME & 6.68mls H2O)

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Lysis of Cultures Cells – General Protocol
 

1) Wash cells twice with ice-cold PBS. Use 3mls (6cm plate) to 5mls (10cm plate) per wash. (For cells in suspension, pellet cells at room temperature, aspirate & discard mediate, add 5-10mls PBS, resuspend with 1ml pipetman, and spin for 2-3min at 1500rpm in refrigerated centrifuge.)

2) Aspirate PBS, let plates drain on an incline of ice for 15", then aspirate off remaining PBS.

3) Add appropriate lysis buffer. Use 0.5-1ml per 10cm plate and 0.25-0.5ml per 6cm plate.

4) Tilt plates back & forth a few times (to ensure even spread of buffer) and place on ice or in refrigerator for 10min.

5) Scrape into pre-chilled microfuge tubes, vortex for 10-20", and place on ice for 10min. (For cells in suspension, add lysis buffer to pelleted cells, triturate with pipetman, and ice for 10min, then transfer to microfuge tube, vortex, and ice for another 10min.)

6) Spin at 14000rpm (~16000xg) for 10min at 4OC.

7) Transfer supernatant to new, pre-chilled microfuge tube.

8) Use 5-10 μl for protein assay, or mix 20 μl with 20 μl 2X sample buffer (or 30 μl with 10 μl 4X sample buffer) for western blotting. Store the rest at -80OC.

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Lysis Buffers and their Applications
RIPA Buffer - For high-stringency lysis (disrupts most protein-protein interactions)

             1%      NP40

          0.5%      NaDOC (deoxycholate)

          0.1%      SDS

        50mM      Tris pH 7.4-7.5

      150mM      NaCl

           10%      glycerol

          1mM      EDTA

Modified RIPA Buffer - Good general-purpose buffer

             1%      NP40

          0.5%      NaDOC

        50mM      Tris pH 7.5

      150mM      NaCl

          5mM      EDTA

NP40 Buffer - Better than mRIPA for some protein-protein interactions but not as clean

             1%      NP40

        50mM      Tris pH 7.5 (Note: can increase to pH 8 to decrease background)

      150mM      NaCl

          5mM      EDTA

Triton X100 Buffer #1 - Use for PAK-Nck and some other co-IPs

             1%      Triton X100

        50mM      Tris pH 7.2

           10%      glycerol

        25mM      b-glycerophosphate

          2mM      EDTA

          2mM      EGTA

Triton X100 Buffer #2 - Use for high-stringency preparation of cytoskeleton (pellet)

             1%      Triton X100

        0.27M      sucrose

        20mM      Tris pH 7.2

        10mM      b-glycerophosphate

          1mM      EDTA

          1mM      EGTA

          0.1%      b-mercaptoethanol

For most applications, use the following protease and phosphatase inhibitors ([final;stock]): NaF (1mM; 1M), Na3VO4 (1mM; 0.1M), AEBSF (100μM; 100mM), benzamidine (5mM; 1M), aprotinin (0.1% of Sigma A6279).

Can also add nitrophenyl phosphate (1mM; 0.1M) and calyculin A (20nM; 100μM) to further inhibit phosphatase activity, if necessary.

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Immunoprecipitation – General Protocol
1) Wash cells twice with PBS and lyse in appropriate lysis buffer;

            Dish size           Wash vol.         Lysis vol.

            1 well               1ml                   125-250μl

            6cm plate         3mls                 250-500μl

10cm plate       5mls                 0.5-1ml

2) Incubate on ice for 10min. Scrape into microfuge tube, vortex for 10”, then ice for an additional 10min.

3) Spin at 4OC for 10min at 14000rpm (Eppendorf microfuge; ~16000xg)

4) Transfer supernatant lysate to new, pre-chilled microfuge tube and remove 5-10μl lysate for protein assay. Discard pellet or, if desired, resuspend in 50-200μl of 1X sample buffer, sonicate and boil.

5) If desired/necessary, pre-clear the lysate by adding 30μl protein A-, protein-G, or protein A/G-sepharose (at 1:1 slurry) and incubating for 30min at 4OC with rocking.