细胞信号转导的技术方法

蛋白磷酸化位点分析及其 功能鉴定
First Military Medical University Chromatograph y Chromatography Chromatography
Phosphopeptide map of HSP27 phosphorylated by PRAK in vitro
检测试剂盒测定底物的磷酸化程度，进 而推出蛋白激酶活性
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较传统激酶活性测定方法的优点
无需接触放射性物质 敏感度高：可以检测到每个磷酸化分子 特异性：利用磷酸化特异性抗体可以进行位点
特异性分析 信噪比高 低背景 系统完整：提供一整套直接从细胞裂解液中测
MAPKAPK2
PRAK
MIX
+
Electrophoresis pH1.9
+
Electrophoresis pH1.9
+பைடு நூலகம்
Electrophoresis pH1.9
First Military Medical University Fold of Activation
T182 is the regulatory phosphorylation site of PRAK
0 5 10 20 30 60 90 120
Time (min)
First Military Medical University Control MKK1(E) MKK7(D)
Effect of individual MAPK pathways on PRAK activity in intact cells
**
hp38 DFGLARHTDD-------------EMTGYVATRWYRAPE 25
hP38 DFGLARQADE-------------EMTGYVATRWYRAPE 25
hp38 DFGLARQADS-------------EMTGYVVTRWYRAPE 25
hp38 DFGLARHADA-------------EMTGYVVTRWYRAPE 25
A
p38-PRAK(182A)
B
+
p38-PRAK(182D)
+
p38-PRAK(wt)
+
Electrophoresis pH 8.9
+- +-+ - + - + - +- +- + -
20 15 10 5 0
p38 HSP27
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DNA重组技术的应用
以免疫共沉淀法获得蛋白激酶 加入该激酶底物和32-ATP，激酶使底
物磷酸化 放射自显影，确定磷酸化条带
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非放射性蛋白激酶活性分析实验流程
以免疫共沉淀法获得蛋白激酶 加入该激酶底物，激酶使底物磷酸化 再加入底物磷酸化特异性抗体 以Phospho Ab-HRP Western 化学发光
试酶活的试剂 节约时间：由几天降到几分钟
First Military Medical University Relative kinase activity
Dynamic processes of p38 activation by LPS in RAW cells
14 12 10 8 6 4 2 0
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细胞信号转导的技术方法
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蛋白激酶磷酸化及其活性测定
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蛋白激酶磷酸化的检测
裂解培养的细胞获得裂解上清 以免疫共沉淀法获得蛋白激酶 SDS-PAGE 再加入蛋白激酶磷酸化特异性抗体 以Phospho Ab-HRP Western 化学发光检
测试剂盒测定蛋白激酶的磷酸化程度。
First Military Medical University
经典蛋白激酶活性分析实验流程
A
B
kDa 97.468.043.9-
29.0-
18.4-
14.3-
-80kDa -47kDa -39kDa
-80kDa -47kDa
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P38 MAP Kinase Pathway
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❖活性诱变体 ❖无活性诱变体 ❖结构与功能的研究
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PCR primer
PCR primer
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The relationship between the members of MAPK family
HSP27 PRAK GST-ATF2 GST-ELK1
First Military Medical University Control Aniso. Arsenite TNF Control Aniso. Arsenite TNF
The major kinases of p38 and p38
JNK2 JNK3 JNK1 p38 p38 p38 p38 ERK1 ERK2 ERK5
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Loop-12(T-Loop) sequence of MAP kinases
MAPK
Dural Phosphorylation Sites L-12 Length
hJNK1 DFGLARTAGTS-----------FMMTPYVVTRYYRAPE 27
hJNK2 DFGLARTACTN-----------FMMTPYVVTRYYRAPE 27
hJNK3 DFGLARTAGTS-----------FMMTPYVVTRYYRAPE 27
hERK1 DFGLARIADPEHDH-------TGFLTEYVATRWYRAPE 31