Edman Degradation Sample Preparation Protocols : Deblocking by pyroglutamdase
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2014-01-27 23:37:45
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사람의 항체 및 천연의 단백질 중 가장 잘 알려진 blocking 형태인 pyroglutamate 의 제거는 다음으로 이어지는 아미노산 서열의 정확한 결정을 위하여 deblocking 과정 중 중요한 전처리 방법을 소개합니다.
Protocol for Deblocking Proteins with pfu Pyroglutamate Aminopeptidase (PGAP)
I. On Membrane.
1. Place PVDF sample in 0.5 ml Eppendorf tube. Wet membrane with 1 µl methanol.
2. BLOCK. Add 200 µl 0.5% polyvinyl pyrrolidone (PVP-360) in 0.1% Acetic acid. Shake for 20 minute at room temperature. Discard supernatant.
3. Rinse. Wash 3 times with 200 µl milli-Q water. Transfer PVDF to a 0.2 ml PCR reaction tube.
4. DIGEST. Add 30 µl of freshly made 1X PGAP buffer consisting of 50 mM Sodium Phosphate, 10 mM DTT, 1 mM EDTA, pH 7.0, recipe to follow:
1. Place PVDF sample in 0.5 ml Eppendorf tube. Wet membrane with 1 µl methanol.
2. BLOCK. Add 200 µl 0.5% polyvinyl pyrrolidone (PVP-360) in 0.1% Acetic acid. Shake for 20 minute at room temperature. Discard supernatant.
3. Rinse. Wash 3 times with 200 µl milli-Q water. Transfer PVDF to a 0.2 ml PCR reaction tube.
4. DIGEST. Add 30 µl of freshly made 1X PGAP buffer consisting of 50 mM Sodium Phosphate, 10 mM DTT, 1 mM EDTA, pH 7.0, recipe to follow:
50mM phosphate buffer:
1.08 g Sodium phosphate monobasic (Na2HPO4*H2O)
3.27 g Sodium phosphate dibasic (Na2HPO4*7 H2O)
q.s. to 200mL with milli-Q water
pH should be 7.0
1.08 g Sodium phosphate monobasic (Na2HPO4*H2O)
3.27 g Sodium phosphate dibasic (Na2HPO4*7 H2O)
q.s. to 200mL with milli-Q water
pH should be 7.0
1X PGAP buffer:
25 mL Phosphate buffer pH 7.0
39 mg DTT
9.3 mg EDTA
25 mL Phosphate buffer pH 7.0
39 mg DTT
9.3 mg EDTA
Dissolve lyophilized enzyme in 100 µl fresh 1X PGAP buffer.
Add 1 mU Pyrococcus furiosus Pyroglumate Aminopeptidase (TaKaRa)*. 10 µl equals 1 mU. Freeze the
remaining enzyme in 10 µl aliquots at -80oC. Incubate sample for 1 hours at 90oC.
Note: Watch reaction to be sure PVDF membrane does not dry out.
1. Discard enzyme solution. Rinse 3 times with 200 µl MQ water.
2. Transfer PVDF to new 0.5 ml eppendorf tube.
3. Dry in speedvac, and store in refrigerator @ 5 degree.
2. Transfer PVDF to new 0.5 ml eppendorf tube.
3. Dry in speedvac, and store in refrigerator @ 5 degree.
II. In Solution.
1. Lyophilize a few µg blocked protein. Use neurotensin or other known PyroGlu-containing protein as a control.
2. REDUCE. Add 100 µl 6M Guan-HCl, 0.2M Tris buffer titrated with glacial acetic acid to pH 8.0, 0.01M DTT (1.54 mg DTT in 1 ml of 6M Guan-HCl 0.2M Tris stock solution). Incubate at 45oC for 1 hour.
3. DESALT. Add protein solution to a Microcon-10 cartridge and spin at 14,000 g for 8 minutes.
4. WASH. Add 100 µl Milli-Q water and spin at 14,000 g until 10 µl remains, approximately 8 minutes. Invert and spin 2 minutes at 1000 g.
5. DIGEST. Add 40 µl of freshly made 1X PGAP buffer consisting of 50 mM Phosphate, 10 mM DTT, 1 mM EDTA, pH 7.0, recipe to follow.
50mM phosphate buffer:
1.08 g Sodium phosphate monobasic (Na2HPO4* H2O)
3.27 g Sodium phosphate dibasic (Na2HPO4*7 H2O)
q.s. to 200mL with milli-Q water
pH should be 7.0
1X PGAP buffer:
25 mL Phosphate buffer pH 7.0
39 mg DTT
9.3 mg EDTA
Dissolve lyophilized enzyme in 100 µl fresh 1X PGAP buffer. Add 1 mU enzyme (TaKaRa)*. 10 µl equals 1 mU. Freeze the remaining enzyme in 10 µl aliquots at -80oC. Incubate sample for 2 hours at 75oC.
25 mL Phosphate buffer pH 7.0
39 mg DTT
9.3 mg EDTA
Dissolve lyophilized enzyme in 100 µl fresh 1X PGAP buffer. Add 1 mU enzyme (TaKaRa)*. 10 µl equals 1 mU. Freeze the remaining enzyme in 10 µl aliquots at -80oC. Incubate sample for 2 hours at 75oC.
6. DESALT. Repeat steps 3 & 4 until 10 µl remain, add 10 µl 2X Tris-Gly reduced sample buffer, invert and spin 2 minutes at 1000 g.
7. Load samples to a 4-20% TG gel, electroblot, and sequence.
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> Pyrococcus furiosus Pyroglutamate Aminopeptidase (TaKaRa TAK7334)
- Note: Stability of the enzyme:
- Lyophilized: room temp for 1 year 100% active
- Dissolved: -20oC for 1 month 100% active, 5oC for 1 month 70% active
> Pyrococcus furiosus Pyroglutamate Aminopeptidase (TaKaRa TAK7334)
- Note: Stability of the enzyme:
- Lyophilized: room temp for 1 year 100% active
- Dissolved: -20oC for 1 month 100% active, 5oC for 1 month 70% active
> REAGENTS:
- Polyvinyl pyrrolidine (PVP-360) FW 360,000
- Tris (hydroxymethyl)aminomethane FW 121.14
- Ethylenediaminetetraacetic acid tetra sodium salt (EDTA)
- C10H12N2O8Na4*4 H2O FW 452.2
- Dithiothreitol (DTT) C4H10O2S2 FW 154.25
- Sodium phosphate monobasic Na2HPO4* H2O FW 120.0
- Sodium phosphate dibasic Na2HPO4*7 H2O FW 142.0
- Polyvinyl pyrrolidine (PVP-360) FW 360,000
- Tris (hydroxymethyl)aminomethane FW 121.14
- Ethylenediaminetetraacetic acid tetra sodium salt (EDTA)
- C10H12N2O8Na4*4 H2O FW 452.2
- Dithiothreitol (DTT) C4H10O2S2 FW 154.25
- Sodium phosphate monobasic Na2HPO4* H2O FW 120.0
- Sodium phosphate dibasic Na2HPO4*7 H2O FW 142.0
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