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小木蟲論壇-學術科研互動平臺 » 生物醫(yī)藥區(qū) » 分子生物 » 綜合其他 » 融合蛋白不掛柱，急！
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甜酒慢品金蟲 (初入文壇)
小木蟲
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[求助]
融合蛋白不掛柱，急！
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大家好，我最近實驗遇到了一個問題要請教大家。 我做的是一個膜蛋白的異源表達，載體用的是Bio rad的pPAL7載體，這個載體有一個特色，就是它的tag在我的蛋白質(zhì)的N端，一旦elution buffer中有氯離子則tag和我的目的蛋白會自動切開。我們通過這個Tag同柱子的親和層析純化我的目的蛋白，然后再加入含有氯離子的溶液得到我的目的蛋白。 tag等電點9.0大小8.3kd，目的蛋白等電點11.7大小5.7kd，融合蛋白等電點9.64（軟件預測），wash buffer是磷酸鈉溶液 pH7.2，融合蛋白在包涵體和可溶性部分均有表達。我每次都搖200ml的菌提取蛋白，可是每次做親和層析都發(fā)現(xiàn)與柱子不結合（可溶性部分和包涵體都試過），望大家指點迷津啊！ |
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2樓2011-09-02 14:45:15
甜酒慢品
金蟲 (初入文壇)
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3樓2011-09-02 14:52:31
4樓2011-09-02 15:27:36
labrat
木蟲 (著名寫手)
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【答案】應助回帖
★ ★ ★ ★ ★
dhd997(金幣+5): good 2011-09-02 18:21:07
甜酒慢品(金幣+5): thank you！ 2011-09-05 10:09:14
dhd997(金幣+5): good 2011-09-02 18:21:07
甜酒慢品(金幣+5): thank you！ 2011-09-05 10:09:14
hi, i don't really understand chinese  , but this is the technical guide generally used for your application:1. Premature elution of cleaved, target protein is observed: Fusion protein: Insert Thr-Ser spacer between Profinity eXact ™ tag and target protein by cloning into pPAL7’s SpeI site Lysis: For maximum yields, do not use lysis buffers and reagents that contain triggering ions, specifically Cl– or F– *Maintain lysate at 4°C prior to loading *Shorten incubation time *Utilize lysis buffer with pH <7.0 Wash: Pre-chill wash buffer to 4°C prior to use *Do not use lysis buffers and reagents that contain triggering ions, specifically Cl– or F–. When preparing wash buffer, substitute sodium acetate (NaOAc) for NaCl; do not use HCl to adjust pH—phosphoric or acetic acid should be used • Utilize wash buffer with pH <7.0 2. Target protein remains uncleaved and/or does not elute completely: Fusion protein: Verify that first amino acid (P1') downstream of the Profinity eXact cleavage site is not proline • If first amino acid of target protein is undesirable for cleavage, insert amino acid spacer (e.g., Thr-Ser) immediately downstream of cleavage site by site-directed mutagenesis or clone into SpeI site of pPAL7 Elution: • Incubate resin with elution buffer for longer time (try up to 1 hour at room temperature or overnight at 4°C) • If performing elution at 4°C, incubate overnight • Increase fluoride concentration in elution buffer • Substitute 10 mM sodium azide for fluoride in elution buffer • Elute with application of second volume of elution buffer to spin column resin 3. Eluate contains significant contaminants: Load:• Dilute lysate for multimeric proteins • Reduce fusion protein load amount to achieve subsaturating level bound to resin • Incubate lysate with resin for up to an hour at 4°C to increase binding capacity of target fusion protein Wash: • Perform additional wash step • Reduce nonspecific, electrostatic binding by increasing ionic concentration of wash buffer; use up to 0.3 M Sodium phosphate, NaOAc, or (NH4)2SO4 (pH 7.2), or amend wash buffer with any of the aforementioned salts. Do not use NaCl • Reduce hydrophobic interactions by decreasing salt concentration of wash buffer • Supplement wash buffer with suitable detergent 4. Fusion protein binds poorly to the resin: Fusion Protein : Proteins with high N-terminal structure may interfere with binding; adding amino acid spacer (i.e., Thr- Ser) between Profinity eXact tag and target protein will improve tag’s accessibility Lysis : • If using urea to help solubilize proteins, use [urea] <4 M; higher urea concentrations reduce binding capacity Load : • Allow lysate to incubate with resin for up to an hour at 4°C or for 30 minutes at room temperature; lysates with fusion proteins >75 kD often benefit from a longer incubation period • When purifying multimeric proteins, avoid saturating resin; diluting lysate may also improve yields Resin :• Resin previously used must be regenerated to remove cleaved Profinity eXact tag; apply three column volumes of 0.1M H3PO4 to resin |
5樓2011-09-02 16:57:40
charlie2800
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6樓2011-09-03 13:56:14
7樓2011-09-30 00:59:24
8樓2011-09-30 01:14:37
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