Multi-Analyte PhosphoELISArray Kits. Brian McNally, Ph.D. Associate Product Manager

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1 Multi-Analyte PhosphoELISArray Kits Brian McNally, Ph.D. Associate Product Manager 1

2 Topics to be Covered Who is SABiosciences? Challenges in Phosphorylation How the PhosphoELISArray Kits Work How YOU Can Use the PhosphoELISArray Kits 2

3 Who is SABiosciences? Located in Frederick, Maryland, USA ~ 30 miles north of the NIH Bethesda campus Founded 10 years ago by NIH scientists Growth through new product development and product line extensions like the ELISArrays 3

4 Systems SABiosciences Research DNA Genomics & Regulation Technologies Sequencing SNP Detection ChIP Methylation SABiosciences Products ChIP-qPCR Assays Methyl-Profiler RNA Discovery Gene Expression Service Core Gene Expression Screening RT 2 PCR Arrays, Oligo GEArrays RT 2 mirna Arrays and Assays Confirmation RT 2 qpcr Assays Protein Detection ELISArray Kits Gene Function Knockdown PhosphoELISArray Kits Protein Function Reporter System sirna Arrays / shrna Plasmids Phenotypes Cignal Reporter System 4

5 Topics to be Covered Who is SABiosciences? Challenges in Phosphorylation How the PhosphoELISArray Kits Work How YOU Can Use the PhosphoELISArray Kits 5

6 Ratio of Phosphorylated to Total Protein Changes P P P P P P P P P P P P 6

7 Challenges Facing Researchers Common Mindset Western Blotting Only one protein at a time. What about others? Which ones? Quantitative Analysis is optional Cost-Effective Multi-Analyte/Multiplex Analysis Format & pricing of Western blots only analyze one protein at a time Multiplex methods require complicated & expensive equipment Who do you believe?! Western Blotting Reagents made by antibody manufacturers 7

8 Western Blotting Screen Four Phosphorylated Proteins $800 - $2000 for the eight primary Abs $125 - $250 for the secondary Abs $75 for Development Solutions $80 for gels $80 for nitrocellulose & filters $90 for molecular weight marker Total: $ $2575 Time: 6 hours using advanced systems, generally covers 2 days 8

9 Topics to be Covered Who is SABiosciences? Challenges in Phosphorylation How the PhosphoELISArray Kits Work How YOU Can Use the PhosphoELISArray Kits 9

10 What is a PhosphoELISArray Kit? Simultaneously profiles multiple proteins Uses convenient & simple ELISA protocol COMPLETE kit with standardized set of Lysis Buffer, Secondary Antibodies, Colorimetric Detection Reagents, Buffers AND Eight different twelve-well strips Each strip is coated with one capture antibody All in one 96-well microtiter plate 10

11 What do you need? Samples: Cell Culture or Tissue Samples Isolation Procedure may require additional equipment Standard Bench Equipment: ELISA Plate Reader 11

12 HOW IT WORKS: Protocol Overview & Data Analysis Secondary Ab Detection Ab HRP HRP HRP HRP Capture Ab Standard ELISArray except cell lysis step. SABiosciences provides a lysis buffer with protease and phosphatase inhibitors. 12

13 Single PhosphoELISArray Layout A Single protein, phosphorylated or total protein (phosphorylated and non-phosphorylated) 8 12 well strips, total of 96 wells All wells contain the same Capture Ab 13

14 Multi PhosphoELISArray Layout Phosphorylated form Total Phospho-Akt Akt Phospho-ERK ERK Phospho-Jun Jun Phospho-p53 p well strips, total of 96 wells All wells in a row contain the same Capture Ab 14

15 Multi PhosphoELISArray Use Sample Concentration Sample Concentration Phospho-Akt Akt Phospho-ERK ERK Phospho-Jun Jun Phospho-p53 Negative Treated Untreated Control p53 Affect of Treatment on Phosphorylation level can be measured 15

16 Beats Western Blotting Screen Four Phosphorylated Proteins $800 - $2000 for the eight primary Abs $125 - $250 for the secondary Abs $75 for Development Solutions $80 for gels $80 for nitrocellulose & filters $90 for molecular weight marker Total: $ $2575 Time: 6 hours using advanced systems, generally covers 2 days ~4.5 Hours and 25-50% of the Cost with Pre-validated Reagents 16

17 Topics to be Covered Who is SABiosciences? Challenges in Phosphorylation How the PhosphoELISArray Kits Work How YOU Can Use the PhosphoELISArray Kits 17

18 Performance Data with Akt Untreated and IGF treated A431 cell lysates were analyzed with PhosphoELISArrays Phosphorylated Akt Kits. A431 cell was cultured in 5% CO2 in DMEM with 10% fetal bovine serum. Cells were then starved for 24 hours after cell confluency reached 70-80%. Cells were then stimulated with100 ng/ml IGF for 30 minutes and lysed 18

19 Performance Data with STAT3 Untreated and IFN treated HeLa cell lysates were analyzed with PhosphoELISArrays Phosphorylated STAT3 Kits. HeLa cell was cultured in 5% CO2 in DMEM with 10% fetal bovine serum. Cells were starved 24 hours when the confluency reached 70-80%. Cells were then stimulated with100 ng/ml IFN for 5 minutes and lysed. 19

20 PhosphoELISArray Summary A solution that provides a fast, robust, scalable and affordable method for determining relative phosphorylation of key regulatory proteins. Profile with the highest levels of sensitivity and linearity possible with screened and validated capture and detection antibodies. No special equipment is required, only the ELISA plate reader already in your lab. 20

21 Ordering Information Pricing: (USD List) Single Analyte PhosphoELISArray FES-######A/T - $449 Multi-Protein PhosphoELISArray FEM-5001H - $499 Storage: Box 1 : 4 C Box 2: -20 C 21

22 Human Multi-Analyte PhosphoELISArrays FEM-5001H FES T FES A FES T FES A FES T FES A FES T FES A FES T FES A FES T FES A FES T FES A FES T FES A FES T FES A PhosphoELISArray Human Cancer Multi-Analyte Kit PhosphoELISArray Human p38 (Total) Single Analyte Kit PhosphoELISArray Human p38 (T180/Y182) Single Analyte Kit PhosphoELISArray Human STAT3 (Total) Single Analyte Kit PhosphoELISArray Human STAT3 (Y705) Single Analyte Kit PhosphoELISArray Human IkBa (Total) Single Analyte Kit PhosphoELISArray Human IkBa (S32) Single Analyte Kit PhosphoELISArray Human Akt (Total) Single Analyte Kit PhosphoELISArray Human Akt (S473) Single Analyte Kit PhosphoELISArray Human Erk1/2 (Total) Single Analyte Kit PhosphoELISArray Human Erk 1/2 (T202/Y204) Single Analyte Kit PhosphoELISArray Human c-jun (Total) Single Analyte Kit PhosphoELISArray Human c-jun (S63) Single Analyte Kit PhosphoELISArray Human p53 (Total) Single Analyte Kit PhosphoELISArray Human p53 (S15) Single Analyte Kit PhosphoELISArray Mouse p38 (Total) Single Analyte Kit PhosphoELISArray Mouse p38 (T180/Y182) Single Analyte Kit PhosphoELISArray Mouse Erk1/2 (Total) Single Analyte Kit PhosphoELISArray Mouse Erk1/2 (T202/Y204) Single Analyte Kit 22

23 Contents of Multi-Analyte PhosphoELISArray FEM-5001H: Human Cancer Array Akt(S473), Akt(Total), Erk1/2(T202/Y204), Erk1/2(Total), c-jun(s63), c-jun(total), p53(s15), p53(total) 23

24 Human Single Arrays FEM-5001H FES T FES A FES T FES A FES T FES A FES T FES A FES T FES A FES T FES A FES T FES A FES T FES A FES T FES A PhosphoELISArray Human Cancer Multi-Analyte Kit PhosphoELISArray Human p38 (Total) Single Analyte Kit PhosphoELISArray Human p38 (T180/Y182) Single Analyte Kit PhosphoELISArray Human STAT3 (Total) Single Analyte Kit PhosphoELISArray Human STAT3 (Y705) Single Analyte Kit PhosphoELISArray Human IkBa (Total) Single Analyte Kit PhosphoELISArray Human IkBa (S32) Single Analyte Kit PhosphoELISArray Human Akt (Total) Single Analyte Kit PhosphoELISArray Human Akt (S473) Single Analyte Kit PhosphoELISArray Human Erk1/2 (Total) Single Analyte Kit PhosphoELISArray Human Erk 1/2 (T202/Y204) Single Analyte Kit PhosphoELISArray Human c-jun (Total) Single Analyte Kit PhosphoELISArray Human c-jun (S63) Single Analyte Kit PhosphoELISArray Human p53 (Total) Single Analyte Kit PhosphoELISArray Human p53 (S15) Single Analyte Kit PhosphoELISArray Mouse p38 (Total) Single Analyte Kit PhosphoELISArray Mouse p38 (T180/Y182) Single Analyte Kit PhosphoELISArray Mouse Erk1/2 (Total) Single Analyte Kit PhosphoELISArray Mouse Erk1/2 (T202/Y204) Single Analyte Kit 24

25 Mouse Single Arrays FEM-5001H FES T FES A FES T FES A FES T FES A FES T FES A FES T FES A FES T FES A FES T FES A FES T FES A FES T FES A PhosphoELISArray Human Cancer Multi-Analyte Kit PhosphoELISArray Human p38 (Total) Single Analyte Kit PhosphoELISArray Human p38 (T180/Y182) Single Analyte Kit PhosphoELISArray Human STAT3 (Total) Single Analyte Kit PhosphoELISArray Human STAT3 (Y705) Single Analyte Kit PhosphoELISArray Human IkBa (Total) Single Analyte Kit PhosphoELISArray Human IkBa (S32) Single Analyte Kit PhosphoELISArray Human Akt (Total) Single Analyte Kit PhosphoELISArray Human Akt (S473) Single Analyte Kit PhosphoELISArray Human Erk1/2 (Total) Single Analyte Kit PhosphoELISArray Human Erk 1/2 (T202/Y204) Single Analyte Kit PhosphoELISArray Human c-jun (Total) Single Analyte Kit PhosphoELISArray Human c-jun (S63) Single Analyte Kit PhosphoELISArray Human p53 (Total) Single Analyte Kit PhosphoELISArray Human p53 (S15) Single Analyte Kit PhosphoELISArray Mouse p38 (Total) Single Analyte Kit PhosphoELISArray Mouse p38 (T180/Y182) Single Analyte Kit PhosphoELISArray Mouse Erk1/2 (Total) Single Analyte Kit PhosphoELISArray Mouse Erk1/2 (T202/Y204) Single Analyte Kit 25

26 PhosphoELISArray Kits Questions? Contact Technical Support 9 AM 6 PM EST Telephone: support@sabiosciences.com Brian McNally, Ph.D. bmcnally@sabiosciences.com 26