匿名使用者
匿名使用者 發問時間: 科學化學 · 5 年前

生物化學paper小段翻譯

大家好~~想拜託幫忙翻譯這兩段

1.Phosphocellulose plates were equilibrated with 100 ul of 70%

Ethyl alcohol followed by washing with 100 ul of Milli-Q water

using a vacuum manifold. The pressure in the system was adjusted

to 15 psi and maintained throughout the experiment. The complete

sample volume (from each well) was transferred to the phosphocellulose

plate and filtered through the same, following which 200 ll of 0.8% phosphoric acid was added for washing. Three washes were done to make sure that the unbound radioactivity was completely removed from the plate. Following this, the plate

was completely dried and 100 ll of Microscint ‘‘O’’ was added to the wells and mixed gently for 2 min on a lab MixMate. The plate was sealed and read in a Top Count radioactivity plate reader.

2.Primary screening for compounds was performed

at three concentrations in duplicate. Serially diluted compound

stocks were prepared in 50 concentration in 100% Me2SO in 96-

well plates. One microliter of compound was added in duplicate

(final concentration of Me2SO was 2%),

然後想請問有人知道vacuum manifold 這個是什麼?如何使用嘛?

麻煩大家了>"<

已更新項目:

A master mix of enzyme, 5x buffer, and water was added to

each well, followed by addition of presonicated lipid mix (1:1 mixture

of PIP2 and PtdSer). A mixture of cold ATP including [r-32-

P]ATP was added to each well such that the final concentration

of the mix was 50 ng of enzyme,

2 個已更新項目:

1 ug of lipid, 3uCi of [r-32P]ATP,

with 25 uM ATP. Primary screening for compounds was performed

at three concentrations in duplicate. Serially diluted compound

stocks were prepared in 50x concentration in 100% Me2SO in 96-

well plates. One microliter of compound was added in duplicate

3 個已更新項目:

(final concentration of Me2SO was 2%), mixed, and kept on ice.

After the reaction was initiated with ATP, the plate was incubated

at 30 C with gentle shaking.Reaction continued for 2 h and was

terminated as previously described.

4 個已更新項目:

The dose–response curves

were generated by serially diluting the stock threefold up to 10

concentrations and adding 1 ll to each well. Final concentrations

were 100, 33.3, 11.1, 3.7, 1.23, 0.41, 0.13, 0.04, 0.015, and

0.005 uM. 這些是這部分的全文 麻煩大家了>

2 個解答

評分
  • 5 年前
    最佳解答

    1. 把磷酸纖維96孔盤(Phosphocellulose plates) 加入70%酒精,把磷酸纖維96孔盤放在孔盤抽真空機 vacuum manifold上,抽真空的壓力設15 psi (整個實驗過程都要維持這樣),然後用100ul Milli-Q水洗剛才的把磷酸纖維96孔盤。

    把全部的sample(從每個well)移到磷酸纖維96孔盤並用同樣方法過濾,然後加入200 ll 0.8% 磷酸洗三次,以確保未結合的放射性物質完全沒殘留在磷酸纖維96孔盤。

    之後把磷酸纖維96孔盤完全dry乾,再於每個well加入100 ll Microscint ‘‘O",把磷酸纖維96孔盤放在實驗室用的MixMate上溫和混勻2分鐘。把磷酸纖維96孔盤封住後用Top Count放射線孔盤讀取機讀取放射線的值。

    2. 用三個濃度並且二重複完成主要篩選化合物的工作。

    在96孔盤製備以100% Me2SO連續稀釋化合物stocks至有效濃度的一半。

    二重複加入1 ul的化合物。(Me2SO最終濃度為2%)

    vacuum manifold: 96孔盤專用的抽真空機

    2015-06-17 14:19:29 補充:

    建議您第二點能貼出全部原文

    2015-06-18 16:51:01 補充:

    更正:

    1.的第一句話: 把磷酸纖維96孔盤(Phosphocellulose plates) 加入"100 ul" 70%酒精

    2.的第二句話: 於96孔盤內用100% Me2SO把要連續稀釋的化合物的stock製備成五十倍濃度。

    取1ul化合物加入二重覆 (Me2SO最終濃度為2%),

    參考資料:
  • 允傑
    Lv 5
    5 年前

    .Phosphocellulose板平衡用100微升70%

    乙醇接著用100微升Milli-Q水

    使用真空歧管。在系統中的壓力調節

    到15 psi和保持整個實驗。完整

    樣品的體積(每孔)轉移到磷酸纖維素

    板,並通過相同的,隨後200 LL的0.8%的磷酸溶液中加入用於洗滌過濾。洗滌三次,都是為確保未結合的放射活性完全從板上移開。在此之後,該板

    完全乾燥和100 LL的的Microscint''O''加入到孔中,並輕輕混合用於在實驗室MixMate混勻儀2分鐘。將板密封並讀取一個頂計數放射性板讀數器。

    進行初步篩選的化合物

    三種不同濃度一式兩份。連續稀釋的化合物

    儲液製備50濃度在100%Me2SO在96

    孔板中。化合物之一微升溶液中加入一式兩份

    (Me2SO的最終濃度為2%),

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