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多種疾病的產生都伴隨著蛋白過度磷酸化的機制。研究磷酸化水平，以確定信號傳導。在不評估總蛋白磷酸化和非磷酸化水平的情況下，研究人員可能會得出錯誤的結論，即一種化合物具有上調或下調磷酸化的作用，而實際上其作用可能是由于該蛋白質表達水平的變化。這種錯誤的分析結果導致對化合物的功能的判定。
HTRF®--均相時間分辨熒光，時間分辨（TRF）和熒光共振能量轉移（FRET）兩大核心技術的碰撞，利用兩個極其穩定的熒光基團，“相遇即發光”的原理，提供了一個簡單（免洗），快速，高通量，高靈敏的方法分析磷酸化蛋白和總蛋白水平。
HTRF分析總蛋白和磷酸化蛋白
本研究進行了總AKT和磷酸化AKT Ser473檢測，尤此提供總蛋白檢測重要性的依據。
實驗方法：所有檢測方法均采用標準的貼壁細胞雙板HTRF檢測方法進行。細胞與化合物在96孔板中孵育，裂解細胞，轉移到兩個不同的檢測板上，一個用于測量磷酸化AKT，另一個用于測量總AKT，并添加HTRF試劑進行檢測。
HEK293細胞用于100000個細胞/孔的所有AKT檢測。細胞與不同濃度的IGF-1孵育10分鐘；已知的蛋白質合成抑制劑環己胺，孵育16小時。數據均以HTRF Ratio值和歸一化值表示。
用IGF-1處理細胞以劑量依賴的方式顯著增加了AKT Ser473磷酸化水平，EC50值為1.06nM。IGF-1也下調了AKT的表達，半抑制濃度值為0.8nM。使用兩種分析數據計算的歸一化值P-AKT/T-AKT顯示，在IGF-1刺激下，磷酸化的AKT與非磷酸化的AKT的比例顯著增加。
環己酰亞胺處理AKT磷酸化水平降低，且AKT表達水平降低，且半抑制濃度值相似。如果沒有進行總AKT檢測，并且只評估磷酸化AKT檢測結果，那么環己酰亞胺可能被錯誤地定義為AKT磷酸化的抑制劑。通過進行兩種分析和將數據表示為歸一化值，可以發現，AKT的磷酸化水平不受環己酰亞胺處理的調節。因此，環己酰亞胺可以被正確地識別為AKT表達的抑制劑，其半抑制濃度值為460nM，但它并不抑制磷酸化。
結合上述實驗結論，可以幫助我們進一步了解同時檢測磷酸化蛋白和總蛋白的重要性，正確并客觀的認定化合物對蛋白磷酸化水平的調節。
相關推薦：
| 名稱 | 規格 | 貨號 |
| 4EBP1, Phospho-T37/46 Kit | 500 tests | 64BP1PEG |
| Alpha-synuclein, Phospho-S129 Kit | 500 tests | 6FSYNPEG |
| Alpha-synuclein, Total Kit | 500 tests | 6FNSYPEG |
| Alpha-Tubulin Housekeeping Kit | 500 tests | 64ATUBPEG |
| ACC, Phospho-S79 Kit | 500 tests* | 64ACCPEG |
| ACC, Total Kit | 500 tests | 64NACPEG |
| AKT, Phospho-S473 Kit | 500 tests* | 64AKSPEG |
| AKT, Phospho-T308 Kit | 500 tests | 64AKTPEG |
| AKT, Total Kit | 500 tests* | 64NKTPEG |
| AKT1, Phospho-S473 Kit | 500 tests | 63ADK078PEG |
| AKT1, Total Kit | 500 tests | 63ADK079PEG |
| AKT2, Phospho-S473 Kit | 500 tests | 63ADK080PEG |
| AKT2, Total Kit | 500 tests | 63ADK081PEG |
| AKT3, Phospho-S473 Kit | 500 tests | 63ADK082PEG |
| AKT3, Total Kit | 500 tests | 63ADK083PEG |
| AMPK, Phospho-T172 Kit | 500 tests* | 64MPKPEG |
| AMPK, Total Kit | 500 tests | 63ADK060PEG |
| Human Androgen Receptor Kit | 500 tests | 64ANDRPEG |
| AP2, Total Kit | 500 tests | 64AP2TPEB |
| ATF2, Phospho-T71 Kit | 500 tests | 63ADK015PEG |
| ATM, Phospho-S1981 Kit | 500 tests | 64ATMS1PEG |
| ATM, Total Kit | 500 tests | 64ATMTPEG |
| BAD, Phospho-S112 Kit | 500 tests* | 64BADPEG |
| Beta Catenin, Phospho-T41/S33/37 Kit | 500 tests | 64CATPEG |
| B-Arrestin 1, Total Kit | 500 tests | 64BAR1TPEB |
| B-Arrestin 2 recruitment Kit | 500 tests* | 62BDBAR2PEB |
| B-arrestin 2, Total Kit | 500 tests | 64BAR2TPEB |
| Beta Catenin, Total Kit | 500 tests | 64NCAPEG |
| BRD2, Total Kit | 500 tests | 64BRD2TPEG |
| BRD3, Total Kit | 500 tests | 64BRD3TPEG |
| BRD4, Total Kit | 500 tests | 64BRD4TPEG |
| BRD7, Total Kit | 500 tests | 64BRD7TPEG |
| BRD9, Total Kit | 500 tests | 64BRD9TPEG |
| BTK, Phospho-Y223 Kit | 500 tests | 63ADK017PEG |
| BTK, Total Kit | 500 tests | 63ADK064PEG |
| CDK1, Total Kit | 500 tests | 64CDK1TPEG |
| CDK2, Phospho-Y15 Kit | 500 tests | 64CDK2Y15PEG |
| CDK2, Total Kit | 500 tests | 64CDK2TPEG |
| CDK4, Total Kit | 500 tests | 64CDK4TPEG |
| CDK6, Total Kit | 500 tests | 64CDK6TPEG |
| CDK7, Total Kit | 500 tests | 64CDK7TPEG |
| CDK9, Total Kit | 500 tests | 64CDK9TPEG |
| CDK12, Total Kit | 500 tests | 64CDK12TPEG |
| Cereblon, Total Kit | 500 tests | 64CRBNPEG |
| CHK1, Phospho-S345 Kit | 500 tests | 64CHK1S5PEG |
| CHK1, Total Kit | 500 tests | 64CHK1TPEG |
| CHK2, Phospho-T68 Kit | 500 tests | 64CHK2T8PEG |
| CHK2, Total Kit | 500 tests | 64CHK2TPEG |
| cJun, Phospho-S63 Kit | 500 tests | 64JUNPEG |
| cRaf, Phospho-S338 Kit | 500 tests | 63ADK018PEG |
| cRaf, Phospho-S43 Kit | 500 tests | 64CRAFS43PEG |
| cRaf, Total Kit | 500 tests | 64CRAFTPEG |
| CREB, Phospho-S133 Kit | 500 tests* | 64CREPEG |
| CREB, Total Kit | 500 tests | 63ADK052PEG |
| Cyclin D1, Phospho-T286 Kit | 500 tests | 64CYCD1T6PEG |
| Cyclin D1, Total Kit | 500 tests | 64CYCD1TPEG |
| c-Src, Phospho-Y419 Kit | 500 tests | 64SRCPEG |
| c-Src, Total Kit | 500 tests | 64NSRPEG |
| EGFR, Phospho-pan Kit | 500 tests | 64HR1PEG |
| EGFR, Phospho-Y1068 Kit | 500 tests | 64EG1PEG |
| EGFR, Total Kit | 500 tests | 64NG1PEG |
| EIF2 alpha, Phospho-S52 Kit | 500 tests | 64EF2PEG |
| EIF2 alpha, Total Kit | 500 tests | 64NEFPEG |
| EIF4E, Phospho-S209 Kit | 500 tests | 64EF4PEG |
| eNOS, Phospho-S1177 Kit | 500 tests | 64ENOSS7PEG |
| eNOS, Total Kit | 500 tests | 64ENOSTPEG |
| ERK, Phospho-T202 /Y204 Advanced Kit | 500 tests* | 64AERPEG |
| ERK, Phospho-T202/Y204 Kit | 500 tests* | 64ERKPEG |
| ERK, Total Kit | 500 tests | 64NRKPEG |
| Estrogen Recepor alpha, Total Kit | 500 tests | 64ERAPEG |
| FAK, Phospho-Y397 Kit | 500 tests | 64FAKPEG |
| FAK, Total Kit | 500 tests | 64NAKPEG |
| FGFR1, Phospho-Y653/654 Kit | 500 tests | 64FGFR1Y6PEG |
| FGFR1, Total Kit | 500 tests | 64FGFR1TPEG |
| FGFR2, Phospho-Y653/654 Kit | 500 tests | 64FGFR2Y6PEG |
| FGFR2, Total Kit | 500 tests | 64FGFR2TPEG |
| FGFR3, Phospho-Y647/648 Kit | 500 tests | 64FGFR3Y6PEG |
| FGFR3, Total Kit | 500 tests | 64FGFR3TPEG |
| FGFR4, Phospho-Y642 Kit | 500 tests | 64FGFR4Y6PEG |
| FGFR4, Total Kit | 500 tests | 64FGFR4TPEG |
| FoxO1, Phospho-S256 Kit | 500 tests | 64FOXPEG |
| FoxO1, Total Kit | 500 tests | 64NFOPEG |
| GSK3 alpha, Phospho-S21 Kit | 500 tests | 64GPAPEG |
| GSK3 alpha, Total Kit | 500 tests | 64GTAPEG |
| GSK3 beta, Phospho-S9 Kit | 500 tests | 64GPBPEG |
| GSK3 beta, Total Kit | 500 tests | 64GTBPEG |
| H2AX, Phospho-S139 Kit | 500 tests | 64H2XPEG |
| HER2, Phospho-Y1221/1222 Kit | 500 tests | 64HR2PEG |
| HER2, Total Kit | 500 tests | 64NR2PEG |
| HER3, Phospho-Y1289 Kit | 500 tests | 64HR3PEG |
| HER3, Total Kit | 500 tests | 64NR3PEG |
| Histone H3, Phospho-S10 Kit | 500 tests | 64HH3PEG |
| Histone H3, Phospho-T3 Kit | 500 tests | 63ADK061PEG |
| Histone H3, Total Kit | 500 tests | 64NH3PEG |
| IKKB, Phospho-S177/181 Kit | 500 tests | 64KKBPEG |
| IKKB, Total Kit | 500 tests | 63ADK097PEG |
| IRAK3, Total Kit | 500 tests | 63ADK101PEG |
| IRAK4, Total Kit | 500 tests | 63ADK108PEG |
| IR beta, Phospho-Y1150 /1151 Kit | 500 tests | 63ADK016PEG |
| IR beta, Total Kit | 500 tests | 63ADK019PEG |
| IRF3, Phospho-S386 Kit | 500 tests | 6FRF3PEG |
| IRF5, Total Kit | 500 tests | 64HIRF5PEG |
| IRS1, Phospho-Ser312 Kit | 500 tests | 64IRSPEG |
| IRS1, Total Kit | 500 tests | 64NRSPEG |
| IRS2, Total Kit | 500 tests | 64IRTPEG |
| Human JAK1, PhosphoTyr1034/1035 Kit | 500 tests | 64JAK1Y10PEY |
| Human JAK1, Total Kit | 500 tests | 64JAK1TPEY |
| Human JAK2, Total Kit | 500 tests | 64JAK2TPEY |
| Human JAK2, Total Kit | 500 tests | 64JAK2TPEY |
| Human JAK3, Total Kit | 500 tests | 64JAK3TPEY |
| JNK, Phospho-T183/Y185 Kit | 500 tests | 64JNKPEG |
| LRRK2, Phospho-S935 Kit | 500 tests | 6FLRKPEG |
| LRRK2, Total Kit | 500 tests | 6FNRKPEG |
| MEK1, Phospho-S218/222 Kit | 500 tests | 64ME1PEG |
| MEK1, Phospho-S298 Kit | 500 tests | 64MEK1S98PEG |
| MEK1, Total Kit | 500 tests | 64NE1PEG |
| MEK1/2, Phospho-S218/222 Kit | 500 tests | 64ME2PEG |
| MKK4, Phospho-S257 Kit | 500 tests | 64MK4CPEG |
| MKK4, Total Kit | 500 tests | 64NK4CPEG |
| MLKL, Phospho-S358 Kit | 500 tests | 64MLKLS8PEG |
| MLKL, Total Kit | 500 tests | 64MLKLTPEG |
| MNK, Phospho-T197/202 Kit | 500 tests | 63ADK022PEG |
| mTOR, Phospho-S2448 Kit | 500 tests | 64TORPEG |
| NDRG1, Phospho-S330 Kit | 500 tests | 63ADK093PEG |
| NDRG1, Phospho-T346 Kit | 500 tests | 63ADK092PEG |
| NDRG1, Total Kit | 500 tests | 63ADK094PEG |
| NFkB, Phospho-S536 Kit | 500 tests | 64NFBPEG |
| NFkB, Total Kit | 500 tests | 64NFTPEG |
| p21, Total Kit | 500 tests | 64HP21PEG |
| P38, Phospho-T180/Y182 Kit | 500 tests | 64P38PEG |
| P53, Phospho-S15 Kit | 500 tests | 64P53PEG |
| P53, Total Kit | 500 tests | 64T53PEG |
| P62/SQSTM1, Phospho-S403 Kit | 500 tests | 64P62S4PEG |
| P62/SQSTM1, Total Kit | 500 tests | 64N62PEG |
| p70S6K, Phospho-T389 Kit | 500 tests | 64S6KPEG |
| PDGFR beta, Phospho-Y751 Kit | 500 tests | 64PDGPEG |
| Phospholamban, Phospho-S16 Kit | 500 tests | 64PLN16PEG |
| Phospholamban, Phospho-T17 Kit | 500 tests | 63ADK074PEG |
| Phospholamban, Total Kit | 500 tests | 63ADK075PEG |
| PLCg1, Phospho-S1177 Kit | 500 tests | 64PLCG1Y3PEG |
| PLCg1, Total Kit | 500 tests | 64PLCG1TPEG |
| PLCg2, Phospho-Y1214 Kit | 500 tests | 64PLCG2Y7PEG |
| PLCg2, Total Kit | 500 tests | 64PLCG2TPEG |
| Human Progesterone Receptor, Total Kit | 500 tests | 64HPRPEG |
| RB, Phospho-S780 Kit | 500 tests | 64RBS780PEG |
| RB, Phospho-S807/811 Kit | 500 tests | 64RB807PEG |
| RB, Total Kit | 500 tests | 64NRBPEG |
| RET, Phospho-pan/Y Kit | 500 tests | 64RETYPEG |
| RET, Total Kit | 500 tests | 64RETTPEG |
| RIPK1, Phospho-S166 Kit | 500 tests | 64RIPK1S1PEG |
| RIPK1, Total Kit | 500 tests | 64RIPK1TPEG |
| RSK1, Phospho-S380 Kit | 500 tests | 64RSK1S0PEG |
| RSK1, Phospho-T359 Kit | 500 tests | 64RSK1T9PEG |
| RSK1, Total Kit | 500 tests | 64RSK1TPEG |
| S6RP, Phospho-S235/236 Kit | 500 tests | 64RP6PEG |
| SHP1, Phospho-Y564 Kit | 500 tests | 64SH1PEG |
| SHP1, Total Kit | 500 tests | 64NH1PEG |
| SHP2, Phospho-Y542 Kit | 500 tests | 64SH2PEG |
| SHP2, Total Kit | 500 tests | 64NH2PEG |
| SLP-76, Phospho-S376 Kit | 500 tests | 63ADK076PEG |
| SLP-76, Phospho-Y145 Kit | 500 tests | 64SLPY5PEG |
| SLP-76, Total Kit | 500 tests | 63ADK077PEG |
| Mouse SLP-76, Phospho-S376 Kit | 500 tests | 63ADK109PEG |
| Mouse SLP-76, Total Kit | 500 tests | 63ADK110PEG |
| SMAD1, Phospho-S463/465 Kit | 500 tests | 63ADK062PEG |
| SMAD1, Total Kit | 500 tests | 63ADK063PEG |
| SMAD2, Phospho-S465/467 Kit | 500 tests | 64SMAD2S5PEG |
| SMAD2, Total Kit | 500 tests | 64SMAD2TPEG |
| SMAD3, Phospho-S423/425 Kit | 500 tests | 63ADK025PEG |
| SMAD3, Total Kit | 500 tests | 64ND3PEG |
| SMAD4, Phospho-T277 Kit | 500 tests | 64SMAD4T7PEG |
| SMAD4, Total Kit | 500 tests | 64SMAD4TPEG |
| STAT1, Phospho-Y701 Kit | 500 tests | 63ADK026PEG |
| STAT1, Total Kit | 500 tests | 63ADK096PEG |
| STAT3, Phospho-Y705 Kit | 500 tests | 62AT3PEG |
| STAT3, Total Kit | 500 tests | 64NT3PEG |
| STAT5, Phospho-Y694 Kit | 500 tests | 64AT5PEG |
| STAT6, Phospho-Y641 Kit | 500 tests | 64AT6PEG |
| STAT6, Total Kit | 500 tests | 64AT6PEG |
| Mouse STING, Phospho-S365 Kit | 500 tests | 64MSTGPEG |
| STING, Phospho-S366 Kit | 500 tests | 64STGPEG |
| STING, Total Kit | 500 tests | 64NTGPEG |
| Mouse STING, Total Kit | 500 tests | 64NMSTGPEG |
| GAPDH Housekeeping Kit | 500 tests | 64GAPDHPEG |
| SYK, Phospho-Y525/526 Kit | 500 tests | 64SYKY525PEG |
| SYK, Total Kit | 500 tests | 64SYKTPEG |
| Tau, Phospho-S202/T205 Kit | 500 tests | 64TS2PEG |
| Tau, Phospho-S356 Kit | 500 tests | 64S356PEG |
| Tau, Phospho-S404 Kit | 500 tests | 64TS4PEG |
| Tau, Phospho-T181 Kit | 500 tests | 64TT181PEG |
| Tau, Total Kit | 500 tests | 64NTAUPEG |
| TBK1, Phospho-S172 Kit | 500 tests | 64TBKPEG |
| TBK1, Total Kit | 500 tests | 64NTBPEG |
| Tie2, Phospho-Y992 Kit | 500 tests | 64TIEY2PEG |
| Ubiquitin, Phospho-S65 Kit | 500 tests | 64UBIS65PEG |
| TYK2, Phospho-Y1054/55 Kit | 500 tests | 64TYK2Y10PEG |
| TYK2, Total Kit | 500 tests | 64TYK2TPEG |
| ULK1, Phospho-S758 Kit | 500 tests | 64ULKPEG |
| VASP, Phospho-S157 Kit | 500 tests | 63ADK066PEG |
| VASP, Phospho-S239 Kit | 500 tests | 63ADK065PEG |
| VASP, Total Kit | 500 tests | 63ADK067PEG |
| VEGFR2, Total Kit | 500 tests | 64VEGFR2PEG |
| VEGFR2, Phospho-Y1175 Kit | 500 tests | 63ADK041PEG |
| YAP, Phospho-S127 Kit | 500 tests | 64YAPPEG |
| YAP, Total Kit | 500 tests | 64YATPEG |
| ZAP-70, Phospho-Y319 Kit | 500 tests | 64ZAPPEG |
| ZAP-70, Total Kit | 500 tests | 64ZATPEG |
| 名稱 | 規格 | 貨號 |
| Phospho-Total protein lysis buffer #1 | 130 mL | 64KL1FDF |
| Phospho-Total protein lysis buffer #2 | 130 mL | 64KL2FDF |
| Phospho-Total protein lysis buffer #3 | 130 mL | 64KL3FDF |
| Phospho-Total protein lysis buffer #4 | 130 mL | 64KL4FDF |
| Phospho-Total protein lysis buffer #5 | 130 mL | 64KL4FDF |
| Phospho-and Total protein blocking reagent | 2ml | 64KB1AAC |
*同靶點有500test和10000test可提供。小編還有AlphaLISA技術磷酸化蛋白和總蛋白檢測試相關試劑盒可提供-。
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