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.
6) Spin at 4OC for 2min at 14000rpm.
7) Transfer lysate, leaving the last 10μl over the pellet (if possible), to another new, pre-chilled microfuge tube containing an appropriate amount of desired antibody. The amount will depend on the antibody and the abundance (total and relative) of the antigen, but a good starting point is 0.5μg antibody / 500μg lysate.
8) Incubate for 1-2hr at 4OC with rocking. Avoid overnight incubationsa.
9) Add 30-40μl protein A-, protein G-, or protein A/G-sepharose and incubate for 30min to 1hr at 4OC with rocking.
10) Collect beads by spinning at either 14000rpm for 10-15” or 5000rpm for 1min (if the latter, spin at 4OC). Carefully aspirate supernatant from beads. Add 1ml lysis bufferb and vortex briefly.
11) Repeat Step #10 2-3 more times.
12) After last wash, aspirate supernatant and remove remainder of wash buffer with fine gauge (>22g) needle. Process for kinase reaction (as indicated) or add 40-80μl 1X sample buffer and boil for 5min.
Notes: a If antigen is in low-abundance, premix antibody and protein (A/G)-beads, in lysis buffer, for 1hr prior to addition to pre-cleared lysates. This increases the avidity of the immunocomplex but does not increase background.
b Some immunoprecipitations will benefit significantly from following the first or second lysis buffer wash with 1-2 high salt washes (e.g. 500mM LiCl/100mM Tris pH8.6) followed by a low-salt wash (e.g. a 1:5 dilution of high-salt wash). Also, if immunoprecipitating for a kinase reaction, the last wash or two should be in the appropriate kinase buffer.
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Immunoprecipitation Kinase Assay – MAPK
1.) Wash cells 2x with ice-cold PBS.
2.) Add RIPA lysis buffer (0.1% SDS, 0.5%DOC, 1.0% NP40, 50mM Tris pH 7.4, 150mM NaCl, 1mM EDTA) containing phosphatase and protease inhibitors (I use PMSF, aprotinin, pepstatin, NaF and Na-orthovanadate). Use 0.5-1.0ml lysis buffer for 10cm plate (depending on cell density), and scale up or down accordingly.
3.) Scrape lysate and transfer to microfuge tube.
4.) Vortex (10sec) and incubate on ice for 10min.
5.) Spin in a microfuge for 10min at 4oC at 14krpm.
6.) Transfer supernatant lysate to new microfuge tube. Freeze at -80oC or proceed directly to IP. At this point you can pre-clear the lysate by incubating with 30μl protein A/G sepharose for 30min at 4oC with rocking, then centrfuging in the cold for 1min at 14krpm and transfering the supernatant lysate to a fresh tube. Depending on the cell type you are using, this may or may not be necessary - however, it never hurts to do it.
7.) Determine protein concentration by whatever method you’re used to.
8.) Typically, I use 150-200μg per immunoprecipitation. You can use less (as little as 50) but the higher amount gives you a good, easily detectable amount of activity.
9.) To the lysate, add 1.5 μg of anti-ERK antibody (Santa Cruz, SC154 (Erk2) or SC93 (Erk1)), and incubate, with rocking, at 4oC for 1hr.
10.) Add 20-30 μl of protein A/G sepharose (50% slurry) and incubate at 4oC for 30min to 1hr.
11.) Prepare kinase buffer (50mM HEPES or Tris pH 7.4, 10mM MgCl2, 10mM MnCl2, 1mM DTT) and reaction mixture, which is 15 μM ATP, 500 μg/ml MBP in kinase buffer.
12.) Wash the immunoprecipitate 3x with RIPA buffer and once with kinase buffer.
13.) Add 40 μl of the reaction mixture, along with 15 μCi of gamma-32P-ATP (3000Ci/mmol, NEN) to the washed beads and mix very gently by tapping the tube (try to avoid getting beads stuck to the side of the tube, above the level of the reaction mixture).
14.) Incubate at 25oC (room temp.) for 25min, mixing occasionally.
15.) Add sample buffer to 1x (I usually add 20 μl of 3x), and boil samples for 3-5min.
16.) Run samples on 12% or 15% gel. Stain gel, dry and expose to film or Phosphorimager screen. I usually load between 20 and 30 μl, and I can see a strong signal overnight. Sample can also be stored, after boiling, at -20oC.
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IPKA – HA-tagged MAPK (a variation & alternate protocol for MAPK IPKA)
1) Transfect cells with GFP and pcDNAI-ERK1-HA plasmids
2) Transfected cells were allowed to adhere to fibronectin-coated dishes for the given time and then stimulated accordingly with growth factor.
3) Wash cells x 2 with ice-cold PBS and then lyse in modified RIPA buffer (50 mM Hepes, pH 7.5, 1% NP-40, 0.5% sodium deoxycholate, 150 mM NaCl, 50 mM NaF, 1 mM sodium vanadate, 1 mM nitrophenylphosphate, 5 mM benzamidine, 0.2 μM calyculin A, 2 mM PMSF, and 10 μg/ml aprotinin).
4) Lyse for 20 min on ice and clarify lysates by centrifugation at 16,000 x g for 10 min at 4 oC
5) Preclear lysates with 30 μl of a 1:1 slurry of protein G-sepharose (Fast-Flow; Pharmacia Biotech, Cat# 17-0618-01) on a rotator at 4 °C for 30 min.
6) Incubate precleared lysates with anti-HA antibody on ice for 2 h.
7) Add 30 μl Protein G beads and incubate on a rotator at 4 °C for 2 h.
8) Wash immunocomplex once with cold lysis buffer.
9) Wash twice with cold wash buffer (0.1 M NaCl, 0.25 M Tris-HCl, pH 7.5.).
10) To washed immunocomplex, add 40 μl of reaction mixture (10 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM DTT, 25 μM ATP, 5 μCi 32P-ATP (Dupont NEN Cat# NEG-002A) and 10 μg myelin basic protein (UBI or GIBCO)) per assay and incubate on a shaking platform at RT for 30 min.
11) Add 13 μl 4 X SDS sample buffer and boil for 5 min to stop the reaction.
12) Separate reaction by SDS PAGE: 15% to analyze incorporation of 32P into myelin basic protein and 10% gel for Western blotting to obtain levels of expressed HA-ERK1.
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