diff --git a/studies/competition co-IP/isa.study.xlsx b/studies/competition co-IP/isa.study.xlsx index 1b28c7e73fc09974bca2c15ccb04c41083c60f81..da4ae314f86adabc82098670a2385dafe41bc8b5 100644 Binary files a/studies/competition co-IP/isa.study.xlsx and b/studies/competition co-IP/isa.study.xlsx differ diff --git a/studies/competition co-IP/protocols/antibodyaffinitypurification.txt b/studies/competition co-IP/protocols/antibodyaffinitypurification.txt new file mode 100644 index 0000000000000000000000000000000000000000..3fd92dd12c1cf04f06057245860ef8d191eace6f --- /dev/null +++ b/studies/competition co-IP/protocols/antibodyaffinitypurification.txt @@ -0,0 +1,36 @@ +Antibody-Affinity-Purification with NHS-activated beads from GE + +Buffers: +1. PBS +2. 1mM HCl +3. 0.1M Tris pH 7.5 +4. 2% Na-Azide +5. 50 mM Tris pH 8, 150 mM NaCl +6. 100 mM glycin pH 2.5, 500 mM NaCl +7. 1.5 M Tris pH 8.8 +8. PBS, 10% glycerol +Preparation of the protein: +9. The protein solution should not contain any amine-containing buffer (Tris/Glycin) +10. Dialyse the protein against 1xPBS or other buffer at pH 7-9 +11. 1 mL slurry can bind 20 mg protein +12. Eg. Couple 4 mg to 200 µL slurry +Preparation of the beads: +13. Pipet slurry with a cut pipet tip to the spin column, let the isoprop flow through and wash the resin with ice cold 1 mM HCl extensively (drops trough) +1. Wash with PBS or your desired buffer +2. Plug the bottom and incubate the resin with the epitope protein for 1 h at RT +3. Collect the flow -through +4. Wash with PBS or your desired buffer (Collect) +5. Quench the remaining NHS-groups with 1 M Tris pH 7.5, for 20 min at RT +6. Collect the flow-through and wash at least 3 times in PBS or your desired buffer +7. Store in that solution with the addition of 0,05% Na-Azide at 4°C +Affinity Purification: +1. Equilibrate the resin in PBS or your desired buffer, wash twice +2. Add the serum 1.4 mL to the resin and incubate (with mixing) at RT for 1-2 h or O/N at 4°C +3. Save the flow-through and wash twice in PBS or your desired buffer +4. Acid prewash in 1 mL 50 mM Tris pH 8, 150 mM NaCl +5. Elute the antibody 2x with 1 mL elution buffer (100 mM glycin oH 2.5) mix 1 min +6. Transfer the eluate directly to 50 µL 1.5 M Tris pH 8.8 +7. Measure the absorbance at 280 nm and pool and dialyse against PBS+10% glycerol +Regeneration: +1. Wash the resin extensively in PBS or your desired buffer +2. Add PBS or your desired buffer containing 0.05% Na-Azide for storage diff --git a/studies/competition co-IP/protocols/antibodycoupling.txt b/studies/competition co-IP/protocols/antibodycoupling.txt index bc1750e147bbefdcca9675186753fcfc62cce3fd..cac96f504e6fccac2163ae13a623f978f72bf73e 100644 --- a/studies/competition co-IP/protocols/antibodycoupling.txt +++ b/studies/competition co-IP/protocols/antibodycoupling.txt @@ -1,2 +1,26 @@ -In-gel digest: -1. \ No newline at end of file +Coupling of antibodies: +1. Prepare a homogenous slurry of the Amintra-ProteinA beads +2. Transfer 100 µL with a wide mouthed tip to a 1.5 mL tube +3. Add 750 µL of 0,1 M Potassium phosphate buffer (KPi), pH 7,5 +4. Spin for 15 sec at 13000 rpm and 4°; carefully remove supernatant +5. Wash 3 x and reconstitute to 750 µL with KPi +6. Use 100 µl beads per 100 µg purified antibody or 50 µl serum +7. Reconstitute 650 µg antibody in 1.6 mL Citrate phosphate buffer, pH 5,0 + 0,1% NP-40 and split in 4 tubes (0.4 mL) add 163 µl (in sum 652 µL) beads to each tube and bring volume up to 1 ml (add 0.437 µL citrate buffer) +8. Rotate >16h at 4°C +9. 4500 g 1 min +10. For every washing step invert few times +11. Wash beads 2x with 1 ml of Citrate phosphate buffer, pH 5,0 + 0,1% NP-40 +12. Resuspend beads in 1 ml of 0.2 M sodium borate buffer, pH 9,0 + 0,1% NP-40 +13. Wash beads 3x with 1 ml of 0.2 M sodium borate buffer, pH 9,0 + 0,1% NP-40 and resuspend beads in 1 mL of this solution +14. Note: Allow DMP to come to RT for 20 min in bag with dessicant prior to usage. Leave dry until just prior to usage! Add 5.3 mg DMP (~ 20 mM) per 1 ml and incubate at RT for 30 min +15. Wash beads 2x with 1 ml of 1M Tris-HCl, pH 7.5 +16. Block the beads in 1 m 1M Tris-HCl, pH 7.5, 1% BSA for 30 min +17. Fill the bead slurry to a volume of ~0.5 ml with 1M Tris-HCl, pH 7.5, 1% BSA, 150 mM NaCl, 0.01% Na-Azide for the storage at 4°C, 0.01% NP40 + +Citrate phosphate buffer, pH 5.0 +Dissolve 2.55 g of Citric acid and 3.65 g sodium phosphate dibasic in 400 ml ddH2O. Adjust the pH to 5.0 with HCl. Bring volume to 500 ml with ddH2O and sterile filter. Store at RT. + +0.5 M Sodium borate, pH 9.0 +Dissolve 15.45 g of boric acid in 400 ml ddH2O and adjust the pH to 9.0 with NaOH pellets. Bring volume to 500 ml with ddH2O and sterile filter. Store at RT. This is a 2.5x stock. + + diff --git a/studies/competition co-IP/protocols/coIP.txt b/studies/competition co-IP/protocols/coIP.txt index bc1750e147bbefdcca9675186753fcfc62cce3fd..ad99ad3732f01a4d98ba751f45348e0bb03efca7 100644 --- a/studies/competition co-IP/protocols/coIP.txt +++ b/studies/competition co-IP/protocols/coIP.txt @@ -1,2 +1,2 @@ -In-gel digest: +Competition co-IP: 1. \ No newline at end of file diff --git a/studies/competition co-IP/protocols/stagetips.txt b/studies/competition co-IP/protocols/stagetips.txt new file mode 100644 index 0000000000000000000000000000000000000000..3d4f73ab1d82e6a078389c866ae1d79f6da5689e --- /dev/null +++ b/studies/competition co-IP/protocols/stagetips.txt @@ -0,0 +1,9 @@ +StageTip desalting: +according to Rappsilber, J., Mann, M. and Ishihama, Y. (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc., 2, 1896–1906. DOI: 10.1038/nprot.2007.261. +1. prepare 200 µL tip with 1 layer 0.8 mm disc Empore C18 +2. condition with 50 µL Sol B (80% acetonitrile, 0.5% acetic acid) +3. equilibrate with 50 µL Sol A (2% acetonitrile, 1% acetic acid) +4. load sample +5. wash 3x with 50 µL Sol A +6. elute 2x with 50 µL Sol B +7. evaporate in speed vac at 2 mbar, 45°C, until dry diff --git a/studies/preExperiment/isa.study.xlsx b/studies/preExperiment/isa.study.xlsx index 2d6c6f0707d8de955afa55907202c7484866eaf7..901abde5c7e11b5d2727eacc7b4af0e3ddcab3d9 100644 Binary files a/studies/preExperiment/isa.study.xlsx and b/studies/preExperiment/isa.study.xlsx differ diff --git a/studies/preExperiment/protocols/.DS_Store b/studies/preExperiment/protocols/.DS_Store new file mode 100644 index 0000000000000000000000000000000000000000..5008ddfcf53c02e82d7eee2e57c38e5672ef89f6 Binary files /dev/null and b/studies/preExperiment/protocols/.DS_Store differ diff --git a/studies/preExperiment/protocols/acetoneprecipitation.txt b/studies/preExperiment/protocols/acetoneprecipitation.txt new file mode 100644 index 0000000000000000000000000000000000000000..a43106ec6c885fb545d6e79bcfbd78d733759d27 --- /dev/null +++ b/studies/preExperiment/protocols/acetoneprecipitation.txt @@ -0,0 +1,8 @@ +aceton precipitation: +1. To 100 µL eluate from the IP add 500 µL acetone +2. precipitate proteins over night at -20°C +3. pellet proteins by centrifugation at 16.000 g, 4°C +4. carefully decant supernatant +5. wash pellet with 500 µL ice cold 80% acetone +6. spin again, but only 5 min +7. carefully decant supernatant, let the pellet air dry for 10-20 min \ No newline at end of file diff --git a/studies/preExperiment/protocols/antibodyaffinitypurification.txt b/studies/preExperiment/protocols/antibodyaffinitypurification.txt new file mode 100644 index 0000000000000000000000000000000000000000..0174c34c8c3fb65955e32b960a84f41ee5b4bd4f --- /dev/null +++ b/studies/preExperiment/protocols/antibodyaffinitypurification.txt @@ -0,0 +1,28 @@ +Antibody-Affinity-Purification with westernblotting + +Buffers: +1. PBS +2. 1mM HCl +3. 0.1M Tris pH 7.5 +4. 2% Na-Azide +5. 50 mM Tris pH 8, 150 mM NaCl +6. 100 mM glycin pH 2.5, 500 mM NaCl +7. 1.5 M Tris pH 8.8 +8. PBS, 10% glycerol +Preparation of the protein: +9. Cast SDS-PAGE 10% with a single large pocket +10. Add 5x Lämmli buffer and cook 1 min at 95°C +11. load as much as possible on the gel +12. run the gel and western blot transfer on a membrane +13. Stain with Ponceau-S and cut out the big protein band, destain, block 30 min with 3% non-fat dry milk in PBS-T +Antibody purification: +1. Put the membrane pieces into a tube +2. add 1 mL of respective serum to the membrane and incubate (with mixing) at RT for 1-2 h or O/N at 4°C +3. Save the flow-through and wash 3x in PBS-T +4. Acid prewash the membranes in 10 mL 50 mM Tris pH 8, 150 mM NaCl +5. Put the membrane in a 2 mL tube and elute the antibody 2x with 0.5 mL elution buffer (100 mM glycin oH 2.5) mix 1 min +6. Transfer the eluate directly to 25 µL 1.5 M Tris pH 8.8 +7. Measure the absorbance at 280 nm and pool and dialyse against PBS+10% glycerol +Regeneration: +1. Wash the resin in PBS-T +2. The dry membrane can be stored at -80°C diff --git a/studies/preExperiment/protocols/antibodycoupling.txt b/studies/preExperiment/protocols/antibodycoupling.txt index bc1750e147bbefdcca9675186753fcfc62cce3fd..cac96f504e6fccac2163ae13a623f978f72bf73e 100644 --- a/studies/preExperiment/protocols/antibodycoupling.txt +++ b/studies/preExperiment/protocols/antibodycoupling.txt @@ -1,2 +1,26 @@ -In-gel digest: -1. \ No newline at end of file +Coupling of antibodies: +1. Prepare a homogenous slurry of the Amintra-ProteinA beads +2. Transfer 100 µL with a wide mouthed tip to a 1.5 mL tube +3. Add 750 µL of 0,1 M Potassium phosphate buffer (KPi), pH 7,5 +4. Spin for 15 sec at 13000 rpm and 4°; carefully remove supernatant +5. Wash 3 x and reconstitute to 750 µL with KPi +6. Use 100 µl beads per 100 µg purified antibody or 50 µl serum +7. Reconstitute 650 µg antibody in 1.6 mL Citrate phosphate buffer, pH 5,0 + 0,1% NP-40 and split in 4 tubes (0.4 mL) add 163 µl (in sum 652 µL) beads to each tube and bring volume up to 1 ml (add 0.437 µL citrate buffer) +8. Rotate >16h at 4°C +9. 4500 g 1 min +10. For every washing step invert few times +11. Wash beads 2x with 1 ml of Citrate phosphate buffer, pH 5,0 + 0,1% NP-40 +12. Resuspend beads in 1 ml of 0.2 M sodium borate buffer, pH 9,0 + 0,1% NP-40 +13. Wash beads 3x with 1 ml of 0.2 M sodium borate buffer, pH 9,0 + 0,1% NP-40 and resuspend beads in 1 mL of this solution +14. Note: Allow DMP to come to RT for 20 min in bag with dessicant prior to usage. Leave dry until just prior to usage! Add 5.3 mg DMP (~ 20 mM) per 1 ml and incubate at RT for 30 min +15. Wash beads 2x with 1 ml of 1M Tris-HCl, pH 7.5 +16. Block the beads in 1 m 1M Tris-HCl, pH 7.5, 1% BSA for 30 min +17. Fill the bead slurry to a volume of ~0.5 ml with 1M Tris-HCl, pH 7.5, 1% BSA, 150 mM NaCl, 0.01% Na-Azide for the storage at 4°C, 0.01% NP40 + +Citrate phosphate buffer, pH 5.0 +Dissolve 2.55 g of Citric acid and 3.65 g sodium phosphate dibasic in 400 ml ddH2O. Adjust the pH to 5.0 with HCl. Bring volume to 500 ml with ddH2O and sterile filter. Store at RT. + +0.5 M Sodium borate, pH 9.0 +Dissolve 15.45 g of boric acid in 400 ml ddH2O and adjust the pH to 9.0 with NaOH pellets. Bring volume to 500 ml with ddH2O and sterile filter. Store at RT. This is a 2.5x stock. + + diff --git a/studies/preExperiment/protocols/coIP.txt b/studies/preExperiment/protocols/coIP.txt index bc1750e147bbefdcca9675186753fcfc62cce3fd..ad99ad3732f01a4d98ba751f45348e0bb03efca7 100644 --- a/studies/preExperiment/protocols/coIP.txt +++ b/studies/preExperiment/protocols/coIP.txt @@ -1,2 +1,2 @@ -In-gel digest: +Competition co-IP: 1. \ No newline at end of file diff --git a/studies/preExperiment/protocols/insolutiondigest.txt b/studies/preExperiment/protocols/insolutiondigest.txt index e1510174f2dabe670e4e60afb7ba73b46c36abbd..0cb29c5f0cda31435940acb251f2121c0cb5c9b5 100644 --- a/studies/preExperiment/protocols/insolutiondigest.txt +++ b/studies/preExperiment/protocols/insolutiondigest.txt @@ -1,2 +1,8 @@ In-solution digest: -1. \ No newline at end of file +1. resuspend the pellet in 20 µL (6 M urea, 2 M thiourea, 50 mM ammonium bicarbonate, 6.5 mM DTT) sonicate in a sanitation bath for 10 min, let then sit on the bench for 20 min +2. add 1 µL 27 mM iodacetamide, incubate in the dark for 20 min +3. add 16 µL of 40 mM ammonium bicarbonate, 2 µL acetonitrile and 3 µL LysC (Wako) +4. incubate >3 h at 37°C +5. add 120 µL of 40 mM ammonium bicarbonate and 5 µL trypsin (0.1 µg/µl) +6. incubate >16 h at 37°C +7. quench digestion with 2 µL acetic acid and 8.5 µL acetonitrile diff --git a/studies/preExperiment/protocols/stagetips.txt b/studies/preExperiment/protocols/stagetips.txt new file mode 100644 index 0000000000000000000000000000000000000000..3d4f73ab1d82e6a078389c866ae1d79f6da5689e --- /dev/null +++ b/studies/preExperiment/protocols/stagetips.txt @@ -0,0 +1,9 @@ +StageTip desalting: +according to Rappsilber, J., Mann, M. and Ishihama, Y. (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc., 2, 1896–1906. DOI: 10.1038/nprot.2007.261. +1. prepare 200 µL tip with 1 layer 0.8 mm disc Empore C18 +2. condition with 50 µL Sol B (80% acetonitrile, 0.5% acetic acid) +3. equilibrate with 50 µL Sol A (2% acetonitrile, 1% acetic acid) +4. load sample +5. wash 3x with 50 µL Sol A +6. elute 2x with 50 µL Sol B +7. evaporate in speed vac at 2 mbar, 45°C, until dry