蛋白质技术

1)grow 20ml cells O/N37℃ dilute 50X into prewarmed LBgrow to 0.6 OD or about 1hr.Induce w/ 2mM IPTG (238mg/0.5l)grow 3hrspin 6k GS3 10'freeze at 70℃ 2)extract cells (from 500ml)in 25 mls.Heintz Buffer ...

1.Prepare 1 ml of packed beads by washing them 4 times in 10 ml of d-H2O in a 15 ml tube.The beads are Act.Ultrogel 22 AcA from IBFand stored in the coldroom.The protein binding site is via glutaralde ...

1.Start a 5 ml overnight culture from a frozen stock (in the -70℃)in the following media: For 100 ml of M9CA media tyrp 10 ml of 10X M9 salts 5 ml of 10% casamino acids 0.2 ml 1M MgSO4 0.01 ml 1M CaC ...

Low (“Low”)Imidazole Buffer 0.5L 100mM Imidizole 3.4g 5% glycerol 25ml 100% glycerol 50mM Tris-HCl (pH 7.9)50ml 0.5M Tris-HCl (pH 7.9) 0.1% Tween-20 0.5M 100% Tween-20 500mM NaCl50ml 5M Na ...

Inoculate 2ml of 5ml o/n culture of either p3133 (empty vector pET11a)or p3134 (dnEBNA-1/Soft)in E.coli BL21 LysS per 0.5L LB ampicillin (grow two 0.5L cultures of each) Incubate ~2hrs 37℃250rpm until ...

Making GST fusion proteins:(07/19/03)ver.1 Grow up 5ml LB with Amp o/n. Add to 45ml LB with Amp 37o shake 2.5- 3 hrstill OD600 0.4-0.8 Put bottles in room temperature water for 10 min to cool do ...

GST Protein Prep.Ver.2 1)Grow 50ml of culture in LB or TB antibiotic o/n at 37℃ shaker. 2)Dilute culture in LB or TB antibiotic 1:10 3)Grow 3hrs at 37℃. 4)Induce culture by adding 0.4 mM IPTG final co ...

Screen of GST-Fusion Protein Expression (pGEX system by Amersham: for check clones for expression of the desired fusion protein prior to large-scale purification) Pick several colonies of E.coli trans ...

Buffers: Lysis Buffer (1 liter) 50 mM NaH2PO4 6.90g NaH2PO4.H2O (MW 137.99g/mol ) 300 mM NaCl(up to 2M)17.54gNaCl(MW 58.44)or 60ml 5M 10 mM Imidazole (up to 100mM)0.68g (MW 68.08 ) 10 mM BME (up to 20 ...

Horvath and RiezmanYeast1994; Gottschling Lab Sample Buffer: 10 ml 0.06M Tris-HClpH 6.8 ...

SDS extraction followed by acetone precipitation – simple extraction protocol that does not require phenol.Recommended start protocol for whole tissue extractions. 1.Grind 1 g of fresh tissue to ...

Buffer A Buffer B &nb ...

Purification of TFIIA from E.coli Linda WarfieldHahn Lab2003 Volumes given are for 2L of cells for each subunit (Toa1 and Toa2). Toa1 and Toa2 subunits are expressed separately in cultures of OD0.6 fo ...

To concentrate proteins for analysis by SDS PAGE: If a small amount of protein is to be precipitated (less than a few micrograms)add Insulin as a carrier protein (10 micrograms of Sigma insulinI-5500p ...

Summary WCE Extracts are prepared from strain SHY282 (TFC4-Flag-EE (HpaI)).TFIIIC complex is first purified by flag affinity: TFC4 contains a single copy of the flag epitope.The flag purified TFIIIC i ...

Horvath and RiezmanYeast1994; Gottschling Lab Sample Buffer: 10 ml 0.06M Tris-HClpH 6.8 0.6 ml 1M Tris 6.8 10% (v/v)glycerol 2 ml 50% glycerol 2% (w/v)SDS 2 ml 10% SDS 5% (v/v)2-mercaptoethanol 0. ...

Adapted from YaffeM.P.and SchatzG.PNAS1984814819-4823. 1.Grow 25mls yeast cells to 5x10E6. 2.Sequentially spin down cells in a 15 ml polypro tube. 3.Wash cells with 1ml ice cold ddH2Otransferrring to ...

Solutions Elution Buffer 50 mM Tris 7.5 5 ml 1M Tris pH 7.5 0.1% SDS 0.1 g SDS 0.1 mg/ml BSA 1.0 mg BSA 0.25 mM EDTA 50 ml 0.5M EDTA 2.5% glycerol 2.5 ml glycerol up to 100 ml with Q For every 10 ml a ...

TGEK Base 50 mM Tris 10% vol Glycerol 1 mM EDTA 100 mM KCl (add for std lysis buffer): PMSF Benzamidine Leupeptin Aprotinin 0.5% NP40 (add for solubilization buffer) 1% NP40 0.1% Triton X100 0.1% SDS ...

The following tips can be used to overcome the most common problems encountered during Western blotting. Smeared Pattern or Distorted Bands Uneven contact between gel and membrane: cassettes used sh ...