Update

studies/competition co-IP/protocols/antibodyaffinitypurification.txt

+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

studies/competition co-IP/protocols/antibodycoupling.txt

-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.
+
+

studies/competition co-IP/protocols/coIP.txt

-In-gel digest:
+Competition co-IP:
 1.
\ No newline at end of file

studies/competition co-IP/protocols/stagetips.txt

+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

studies/preExperiment/protocols/acetoneprecipitation.txt

+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

studies/preExperiment/protocols/antibodyaffinitypurification.txt

+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

studies/preExperiment/protocols/antibodycoupling.txt

-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.
+
+

studies/preExperiment/protocols/coIP.txt

-In-gel digest:
+Competition co-IP:
 1.
\ No newline at end of file

studies/preExperiment/protocols/insolutiondigest.txt

 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

studies/preExperiment/protocols/stagetips.txt

+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