i******m 发帖数: 495 | 1 要做一个ELISA检测细胞膜上一个蛋白的phosphorylation. 按照protocol应该把细胞
lysis以后做sonication, 但是实验室没有sonicator, 有什么什么其他替代方法呢?
Freeze-thaw会不会把磷酸化给弄没了?
thank you |
i******m 发帖数: 495 | 2 upup
【在 i******m 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 要做一个ELISA检测细胞膜上一个蛋白的phosphorylation. 按照protocol应该把细胞 : lysis以后做sonication, 但是实验室没有sonicator, 有什么什么其他替代方法呢? : Freeze-thaw会不会把磷酸化给弄没了? : thank you
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j*****a 发帖数: 658 | 3 Don't quite understand your question.... You want to detect the
phosphorylation of a protein, why not use western? Even if you do ELISA to
see p-protein, no need to sonicate....RIPA is strong enough to get all your
cytosolic and nuclear protein out. Sonication mostly is used to break down
chromatin, DNA or chop down plasma membrane to release cytoplasma membrane
protein....If I am wrong, please someone corrects me.. So I think in your
case, you probably don't need sonication at all.
Also, freeze-thaw will break your plasma membrane because of ice. There is
no reason it hurts your protein phosphorylation. I think...
You sounds like you want to kinase assay, or something.
【在 i******m 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 要做一个ELISA检测细胞膜上一个蛋白的phosphorylation. 按照protocol应该把细胞 : lysis以后做sonication, 但是实验室没有sonicator, 有什么什么其他替代方法呢? : Freeze-thaw会不会把磷酸化给弄没了? : thank you
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y******8 发帖数: 1764 | 4 "膜上一个蛋白". I don't think you can easily get away with a sonicator. Just
ask other labs around. |
i******m 发帖数: 495 | 5 Thank you for your advice.
用ELISA而不用WB是因为ELISA有现成的KIT,不用OPTIMIZE条件。而且ELISA快, 半天
就能出结果。我担心的是如果不用SONICATION的话, 会不会样品很STICKY,不容易准
确量取,各个REPLICATE之间的差异太大。 除了FREEZE THAW,还有别的办法么
to
your
【在 j*****a 的大作中提到】![](/moin_static193/solenoid/img/up.png) : Don't quite understand your question.... You want to detect the : phosphorylation of a protein, why not use western? Even if you do ELISA to : see p-protein, no need to sonicate....RIPA is strong enough to get all your : cytosolic and nuclear protein out. Sonication mostly is used to break down : chromatin, DNA or chop down plasma membrane to release cytoplasma membrane : protein....If I am wrong, please someone corrects me.. So I think in your : case, you probably don't need sonication at all. : Also, freeze-thaw will break your plasma membrane because of ice. There is : no reason it hurts your protein phosphorylation. I think... : You sounds like you want to kinase assay, or something.
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i******m 发帖数: 495 | 6 upup
【在 i******m 的大作中提到】![](/moin_static193/solenoid/img/up.png) : Thank you for your advice. : 用ELISA而不用WB是因为ELISA有现成的KIT,不用OPTIMIZE条件。而且ELISA快, 半天 : 就能出结果。我担心的是如果不用SONICATION的话, 会不会样品很STICKY,不容易准 : 确量取,各个REPLICATE之间的差异太大。 除了FREEZE THAW,还有别的办法么 : : to : your
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Z******5 发帖数: 435 | 7 一直不明白为啥western的样品煮了后都不影响抗体的结合,而ELISA的样品就不能煮。 |
r******y 发帖数: 21907 | 8 我也在做plasma membrane protein的phosphorylation,不过我是植物蛋白,加triton
x-
100或者sds让membrane protein soluble,然后centrifugation 取上清,sonication
是打
碎细胞膜吧,对膜蛋白不好吧。ELISA你用的什么抗体?我估计要用phosphotyrisine
threonine的
antibodies·做Western
【在 i******m 的大作中提到】![](/moin_static193/solenoid/img/up.png) : upup
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i******m 发帖数: 495 | 9 嗯。离心一下应该是个不错的主意。我用的是elisa kit
一个抗体precoat在板子上,另一个抗体来识别。都是phospho specific抗体
triton
sonication
【在 r******y 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 我也在做plasma membrane protein的phosphorylation,不过我是植物蛋白,加triton : x- : 100或者sds让membrane protein soluble,然后centrifugation 取上清,sonication : 是打 : 碎细胞膜吧,对膜蛋白不好吧。ELISA你用的什么抗体?我估计要用phosphotyrisine : threonine的 : antibodies·做Western
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r******y 发帖数: 21907 | 10 哦,那是动物上的抗体了,植物都没有。):
你用什么分离蛋白的?加了sds了?我们是先用1%的SDS再稀释到0.1% final [] no
more than
0.1%, but between 0.05-0.1%
【在 i******m 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 嗯。离心一下应该是个不错的主意。我用的是elisa kit : 一个抗体precoat在板子上,另一个抗体来识别。都是phospho specific抗体 : : triton : sonication
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w***e 发帖数: 269 | 11 找一个最细最小的针头和针管。500uL 或者1mL 都可以。每个样品抽吸吹打15-20下。样品就不会很
粘稠了。 |
y*****1 发帖数: 73 | 12 Don't know much about ELISA, maybe you can chemically lyse the cell. |
y********g 发帖数: 782 | 13 我做CHIP用CHIP kit,里面切chromatin可以用一种enzyme代替sonication,所以我想
这种enzyme应该可以,因为CHIP是用beads来拉蛋白和DNA的,所以我猜这种enzyme应该
可以分开蛋白~~见B2,可以只买enzyme。http://www.biotechniques.com/multimedia/archive/00054/chip-it_express_manu_54059a.pdf |
q*******8 发帖数: 768 | 14 LZ做的什么细胞啊。。。我觉得经济允许的话你就FREEZE THAW试试呗。我做的INSECT
CELL不用SONICATE,不用FREEZE THAW的话是很粘稠的,加SDS吹打都没有用 |