【讨论帖】酵母双杂交讨论区

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标题:【讨论帖】酵母双杂交讨论区

北国毛毛雪[使用道具]
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发表于 2013-5-17 14:21 资料个人空间短消息加为好友

小中大

本人已完成酵母双杂交实验，欲与同道们交流各自的经验，不知大家有兴趣吗？

王蜜蜜[使用道具]
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发表于 2013-5-17 14:44 资料个人空间短消息加为好友

小中大

相关疾病：
眼疾病
谢谢！
我是新手却要涉及此行，请问这样的实验从技术难度上来说可行吗（对于新手）？
某一眼科疾病的基因的蛋白功能未明，但是在全身各处都有表达，只是在眼内是全部exon都表达，在其它组织有些exon没有表达，但是在脑和睾丸内的该基因表达量比在眼内表达要多很多，所以我想在脑cDNA库里寻找和该基因有interaction的蛋白，就用酵母二杂交，请问在技术上讲难点在哪里？具体应该如何开始准备？谢谢

lixi559[使用道具]
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发表于 2013-5-17 14:44 资料个人空间短消息加为好友

小中大

从你谈的情况看，在技术上应该没有太大的难题。其实酵母双杂交从本质上看，技术操作并不难，只是工作量特别大。但对你来说，作为一个新手，还牵涉到一些基本的分子生物学的实验技能问题，可能开始作实验时无从下手，但一旦进入状态后，后期工作就一帆风顺了。所以作为新手的你来说，可以先掌握好一些分子生物学的基本实验，比如说是基因的一般性操作，然后再着手酵母双杂交。这样对你以后的酵母双杂交实验就会受益莫大。我就是象你这样起步的。
另外在选择铒蛋白方面，要大胆一些，不要谨小慎微，前怕狼后怕虎。一般的蛋白都可以用来酵母双杂交实验的。但你在实验前还是先作好酵母细胞毒性实验和自激活实验，以及蛋白表达的western blot，如果都OK，就放心大胆地去作吧！
你会成功的！

王蜜蜜[使用道具]
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发表于 2013-5-17 14:47 资料个人空间短消息加为好友

小中大

谢谢楼主指点，本人对于蛋白的方面实在是不懂，对于基本的分生实验技术应该没有大问题，现在我是在设计下一个课题，因为想在脑C DNA里钓一钓作用蛋白，所以向您请教了。
时间对于我来说不成问题，我还有三年多呢，呵呵。
其次，在准备Y2H之前，除了准备cDNA文库和订购kit之外，其它需要特别什么东西吗？能告诉我所涉及的特别的机器和材料吗？

lixi559[使用道具]
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发表于 2013-5-17 14:48 资料个人空间短消息加为好友

小中大

很高兴见到你能掌握一些基本的实验，我想你要是作的话，一定能成功的。
至于器材的话，你不必需要特殊的东西，因为你既然能作你所说的那些实验的话，作Y2H已经足够了。
铒蛋白的细胞毒性实验和自激活实验是必须要作的，这是你实验的入口，否则你无法下一步的实验。具体方法可以参考说明书进行，如你没有的话，可以到clontech网站上去DOWN一个来，都是免费的。
试剂和质粒等实验用品，我建议你用clontech的和sigma的，比较好用，而且易于掌握，重复性也好。cDNA文库和kit在这两家里都有现成的，不过要提前预定，一般到货要一个月左右的时间，所以你要提前准备。
至于具体要什么，你可以看一看说明书，非常简单。
如果你有时间的话，可以少买KIT，自已作准备。如果你老板有钱的话，或者不想花太多的时间的话，就全部买现成的KIT，能帮你节省下很多时间，而且实验的成功率也高，这一点对你建立信心特别重要。
最后祝你成功！

lixi559[使用道具]
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发表于 2013-5-17 14:48 资料个人空间短消息加为好友

小中大

clontech 的网址为：cuturl('www.clontech.com.')

lixi559[使用道具]
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发表于 2013-5-17 14:49 资料个人空间短消息加为好友

小中大

请问以下的英文应该是。。？
酵母细胞毒性实验和自激活实验，蛋白表达的western blot。
能稍解释以下为什么这些是Y2H前必须要做的前提吗？谢谢

vivian4123[使用道具]
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发表于 2013-5-17 14:49 资料个人空间短消息加为好友

小中大

毒性是toxic,自激活autoactivation。一定要做细胞毒性实验和自激活实验，不做诱饵的毒性实验，如果bait转入酵母对细胞有毒，导致细胞死亡，你还筛什么库；不做自激活实验，如果单独bait就能使报告基因表达，也就是你还没筛库呢，酵母就可变蓝，你还怎么筛库？
希望斑竹加分，我是个新手！

王蜜蜜[使用道具]
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发表于 2013-5-17 14:50 资料个人空间短消息加为好友

小中大

QUOTE:

原帖由 vivian4123 于 2013-5-17 14:49 发表 bbcodeurl('http://bbs.antpedia.com/images/common/back.gif', '%s')
毒性是toxic,自激活autoactivation。一定要做细胞毒性实验和自激活实验，不做诱饵的毒性实验，如果bait转入酵母对细胞有毒，导致细胞死亡，你还筛什么库；不做自激活实验，如果单独bait就能使报告基因表达，也就是你还没筛库呢，酵母 ...

看了一个protocol：
1. Clone the target DNA encoding the protein to be used as bait into the polylinker of a LexA fusion vector (e.g., pMW101 or pMW103) to synthesize an in-frame fusion to LexA. Ensure that a translational stop sequence is present at the carboxyl terminus of the desired bait sequence. The resulting plasmid is referred to as pBait.
2. Set up a series of transformations of the EGY48 lexAop-LEU2 selection strain of yeast using the following combinations of LexA fusion and lexAop-lacZ reporter plasmids:
a. pBait + pMW112 (test for activation)
b. pSH17-4 + pMW112 (positive control for activation)
c. pRFHM1 + pMW112 (negative control for activation)
d. pBait + pJK101 (test for repression/DNA binding)
e. pRFHM1 + pJK101 (positive control for repression)
f. pJK101 alone (negative control for repression)
3. Plate each transformation mixture on selective dropout plates: CM(Glu)-Ura-His (for plasmid combinations a-e) or CM(Glu)-Ura (for plasmid combination f), as appropriate. Incubate the plates for 2-3 days at 30°C to select for transformed yeast colonies that contain the plasmids.
4. Make a master plate of transformants, from which specific colonies can be assayed for the phenotype of activation of lacZ and LEU2 reporters as described in Steps 5-9.
Steps 5-9 are used to test the bait-LexA fusion protein for transcriptional activity and to demonstrate that the fusion of the bait does not affect LexA DNA-binding activity. Several independent colonies are assayed for each combination of plasmids transformed in Step 2.
5. From each transformation a - f (from Step 2), use sterile, standard flat-edged toothpicks to pick approx. 8 colonies.Touch a clean toothpick to the colony to pick up cells, and restreak them as a 1-cm-long streak in a grid on a fresh CM(Glu)-Ura-His or CM(Glu)-Ura plate. As many as 60-80 streaks can generally be grown on a single plate. Incubate the plates overnight at 30°C.
6. On the second day, restreak from the two master plates to each of the following:
Transformations a - f: Streak onto CM(Glu, X-gal)-Ura and onto CM(Gal, X-gal)-UraTransformations a - c: Streak onto CM(Glu)-Ura-His-Leu and onto CM(Gal)-Ura-His-Leu
Only uracil is dropped out (histidine is present) from the X-gal plates, to allow side-by-side comparison of the JK101 plasmid-only transformation ( f ) with ( d,e ) . Lack of selection for the LexA-fused plasmid does not notably affect transcriptional activation over the period of this assay.
7. Incubate the plates for up to 4 days at 30°C.
8. Assay for repression and activation activities:
a. For repression, observe the X-gal phenotype at approx. 12-24 hours after streaking.
b. For activation, observe the X-gal phenotype between 18 and 72 hours after streaking.
c. Observe the Leu2 phenotype between 48 and 96 hours. The expected results for a well-behaved bait are summarized below.
? Optimally, at 12-24 hours after streaking to CM(Gal, X-gal)-Ura, the d + e transformants should be discernibly lighter in color than f.
? At 48 hours after streaking to CM(Glu, X-gal)-Ura, d transformants should be bright blue, c should be white, and a
should be white or very light blue.

王蜜蜜[使用道具]
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发表于 2013-5-17 14:50 资料个人空间短消息加为好友

小中大

? At 48 hours after streaking, b transformants should be as well grown on CM(Glu)-Ura-His-Leu or CM(Gal)-Ura-His-
Leu as on a CM(Glu)-Ura-His master plate, whereas a and c should show no growth.
? Ideally, a transformants will still display no apparent growth at 96 hours after streaking.
9. Select the appropriate candidate colonies, based on the results of the repression and activation assays.
10. On the master plate, mark the colonies that are to be assayed for protein expression. Use the colony that has been shown to express bait appropriately as the founder to grow a culture for transformation of a library
11. Analyze at least two primary transformants for each novel bait construct. Include two transformants as positive controls for protein expression (e.g., pRFHMI).
a. Use a sterile toothpick to pick colonies from the CM(Glu)-Ura-His master plate into CM(Glu)-Ura-His liquid medium.
b. Grow the cultures overnight on a roller drum or other shaker at 30°C.
c. In the morning, dilute the saturated cultures into fresh tubes containing 3-5 ml of CM(Glu)-Ura-His, with a starting density of OD600 of approx. 0.15. Incubate the cultures for 4-6 hours at 30°C until the optical density has doubled approx. twice (OD600 approx. 0.45-0.7).
12. Transfer 1.5 ml of each culture to a microfuge tube, and centrifuge the cells at maximum speed for 3-5 minutes in a microfuge. The volume of the visible pellet should be 2-5 μl. Carefully decant or aspirate the supernatant.
13. Add 50 μl of 2x SDS gel-loading buffer to the tube, and vortex the tube rapidly to resuspend the pellet. Immediately place the tube either on dry ice or in a dry ice/ethanol bath.
14. Transfer the samples from the dry ice or -70°C directly to 100°C and boil them for 5 minutes.
15. Chill the samples on ice and centrifuge them at maximum speed for 5-30 seconds in a microfuge to pellet large cell debris. Load 20-50 μl into each lane of a SDS-polyacrylamide gel.
16. Run the gel and analyze the products to determine whether bait protein of the expected size is expressed at reasonable levels.
17. To anticipate and forestall potential problems, analyze the lysates of yeast containing LexA-fused baits by immunoblotting.

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