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 (2013-5-17 14:50:24)
? 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.
最新回复
王蜜蜜 (2013-5-17 14:44:18)
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 (2013-5-17 14:44:41)
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 (2013-5-17 14:47:42)
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 (2013-5-17 14:48:11)
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 (2013-5-17 14:48:33)
clontech 的网址为：cuturl('www.clontech.com.')
lixi559 (2013-5-17 14:49:15)
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 (2013-5-17 14:49:33)
希望斑竹加分，我是个新手！
王蜜蜜 (2013-5-17 14:50:04)
QUOTE:
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 (2013-5-17 14:50:24)
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.
IAM007 (2013-5-17 14:50:52)
你要用作筛库的蛋白是在细胞膜外还是在细胞内，如果你要筛的蛋白是在膜外，那么你还要考虑一下你的Bait能否在酵母中入核，如果不能入核，就不能使报告基因表达，这样无论你怎么筛也筛不出来，所以通常要用胞内分子或蛋白的胞内域去筛。以膜外分子筛库成功率极低！
这些都是我的经验和血的教训，望斑竹再加点分！
王蜜蜜 (2013-5-17 14:51:13)
QUOTE:
谢谢！你指的是bait要在细胞内吗？
我想做的蛋白是在细胞内的，但是我只是想在其表达较多的组织cDAN库里去找有没有和这个蛋白相互作用的其它蛋白，说不定也是未知功能的，更不能确定其是在胞内或胞外了。
IAM007 (2013-5-17 14:51:59)
我指的是bait最好在细胞内,既然你想做的蛋白是在细胞内的，那你就准备开始受苦吧,开始筛库吧!还有你要做的蛋白是转录因子或其类的吗,如果是的话,有可能会自激活,因为转录因子一般都有DNA结合结构域和转录激活结构域,这样的话没有蛋白与它interaction它也有可能会使报告基因表达,即自激活。
王蜜蜜 (2013-5-17 14:52:33)
QUOTE:
我要做的bait蛋白目前功能不明，只是其N端与the regulator of chromatin condensation (RCC1)有同源性。有人已经在眼内组织的cDNA库里找到了2个相互作用的新蛋白。
IAM007 (2013-5-17 14:53:04)
王蜜蜜 (2013-5-17 14:53:46)
=====================================
能否讲讲gal4系统和lexA系统的适应证和优缺点？
王蜜蜜 (2013-5-17 14:55:06)
我刚找到humana press的一本书，edited by Paul N.MacDonald《Two Hybrid System-Methods and Protocols》,1.8M，如有哪位需要，请PM我。（勿需在此回贴）
u76mp (2013-5-17 14:56:10)
如不行的话，再想别的办法。
u76mp (2013-5-17 14:56:26)
u76mp (2013-5-17 14:56:47)
Y2H的优点就是可能找到一些未知的蛋白，这也是它具有一定的优势所在。同时在作这个实验的时候，在找到蛋白或新蛋白的同时，你还可以直接得到这个蛋白的DNA序列，有的甚至于是它的全长序列！这也是一个具大的收获。比起你用RT-PCR来得到一个蛋白来说更加具有诱惑力。
dongdongqiang (2013-5-17 14:57:12)
我用的是ｃｌｏｎｔｅｃｈ ｍａｔｃｈｍａｋｅｒｓｙｓｔｅｍ ３。
ｂａｉｔ克隆到ＢＤ载体后，经检验无自激活，对酵母无毒性。我用
ｍａｔｉｎｇ方法筛选一个人脑文库。用SD/-leu-Ada-Trp-His培养基筛选后得到的阳性克隆，经分离酵母质粒后，重新转化Ecoli,然后得到纯的酵母ＡＤ质粒。重新用此ＡＤ质粒分别与pGBKT7（空BD载体，阴性对照）和pGBKT7-bait共转化酵母，发现阴性对照SD/-leu/Trp-His板上丛集性出现的小菌落，而与pGBKT7-bait共转化的在３缺培养基上出现少而大的酵母菌落。这样的结果能否说明得到的ＡＤ克隆为假阳性或真阳性？如果ＡＤ克隆本身为转录活化因子，我是否应该直接用单一的ＡＤ质粒转化酵母继而在SD/-leu-his筛选？谢谢！
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