Tu M2-PK Detection

COLORECTAL CANCERPLASMA, TUMOR TISSUE,

NORMAL TISSUE

2013.04.22

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

Tumor M2-PK - EDTA Plasma test

Tumor M2-PK Antibodies – Tissues test

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. 

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.

Ref.

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

SRSF2 cloning study

New primer designed:

Product size: 662 bp

PCR results

SRSF2 –ex tag gradient [50-70 °C]

Ex Tag 0.3buffer Ex tag 5dNTP 4cDNA 5Primer F 1

R 1DW 33.75Total 50

96  °C : 96  °C : grad °C : 72  °C : 20  °C3min : 30 sec :30 sec : 5 min : --

35 cycles

PCR results

F-Tag 0.3buffer F-tag 5dNTP 1cDNA 5Primer F 1

R 1DW 36.7Total 50

96  °C : 96  °C : 58°C : 72  °C : 20  °C3min : 30 sec :30 sec : 5 min : --

35 cycles

SRSF2 – f-tag 58 °C annealing temp

IM9 ladder

500 bp

1000 bp

F-Tag 0.3buffer F-tag 5dNTP 1cDNA 5Primer F 1

R 1DW 36.7Total 50

96  °C : 96  °C : 60°C : 72  °C : 20  °C3min : 30 sec :30 sec : 5 min : --

35 cycles

PCR results

SRSF2 – f-tag 60 °C annealing temp

K562 Hela IM9 ladder