Tu M2-PK Detection

1. Tu M2-PK Detection COLORECTAL CANCER PLASMA, TUMOR TISSUE, NORMAL TISSUE 2013.04.22

2. Plan for Tu M2-PK study in colorectal cancer • Materials: 370 patients who have plasma, tumor tissue and correspondent normal tissue. • Methods: • For plasma: ELISA (Schebo. Biotech Company) http://www.schebo.com/english/ScheBo_Tumor_M2-PK_EDTA_Plasma_Test_2.php • For tissue: Immunohistochemistry (Schebo. Biotech Company) http://www.schebo.com/english/ScheBo_Tumor_M2-PK_Antibodies.php

3. Tumor M2-PK - EDTA Plasma test

4. Tumor M2-PK Antibodies – Tissues test

5. M2-PK staining of a human colon carcinoma with the anti-M2-PK antibody clone DF4 Immunohistochemical Staining Immunohistology, also known as "immunohistochemistry" is the use of antibodies to stain histological sections. 

6. Immunohistochemistry Vs immunofluorescence • This technique, immunohistochemistry (IHC), is methodologically identical to immunofluorescence (IF), which had been in use since 1941 (Coons et al., 1941). The increasing popularity of IHC over IF is due to four main factors. First, IF requires special equipment, a fluorescence microscope and a designated darkened area. IHC, on the other hand, can be performed using a standard light microscope. Second, IF requires a dark field, so it is not possible to examine fluorescence patterns and cell and tissue morphology simultaneously. IHC utilizes a light microscope for visualization, so antigen localization and cell and tissue morphology can be observed at the same time. Third, fluorochrome-based signals last only 3 to 10 days, depending on the signal intensity and storage conditions. The colored precipitate in IHC staining, particularly with 3,3¢-diamnobenzidine (Table 21.4.2), remains vibrant for years, and provides an excellent permanent record. Fourth, IF fluorochromes are light sensitive, so special care must be taken throughout the staining procedure to minimize loss of signal. The enzymes and most substrates used in IHC, on the other hand, are relatively light insensitive, and therefore the staining procedure can easily be performed on the open benchtop without any unusual precautions. The disadvantage of some IHC chromogens is their known carcinogenic effects, but using gloves when handling these reagents is an adequate safety precaution. The availability of a variety of enzyme-conjugated antibodies and colored substrates (see Table 21.4.2) has made IHC more convenient and accessible to many laboratories.

7. Ref. • http://www.metabolic-database.com/html/pyruvate_kinase_isoenzymes.html

8. SRSF2 cloning study New primer designed: Product size: 662 bp

9. PCR results • SRSF2 –ex tag gradient [50-70 °C] Ex Tag 0.3 buffer Ex tag 5 dNTP 4 cDNA 5 Primer F 1 R 1 DW 33.75 Total 50 35 cycles 96  °C : 96  °C : grad °C : 72  °C : 20  °C 3min : 30 sec :30 sec : 5 min : --

10. PCR results • SRSF2 – f-tag 58 °C annealing temp IM9 ladder F-Tag 0.3 buffer F-tag 5 dNTP 1 cDNA 5 Primer F 1 R 1 DW 36.7 Total 50 35 cycles 96  °C : 96  °C : 58°C : 72  °C : 20  °C 3min : 30 sec :30 sec : 5 min : -- 1000 bp 500 bp

11. PCR results • SRSF2 – f-tag 60 °C annealing temp F-Tag 0.3 buffer F-tag 5 dNTP 1 cDNA 5 Primer F 1 R 1 DW 36.7 Total 50 K562 Hela IM9 ladder 35 cycles 96  °C : 96  °C : 60°C : 72  °C : 20  °C 3min : 30 sec :30 sec : 5 min : --