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2.2.16.1 Quantitative assay of B-galactosidase activity by spectrophotometry

Quantitative analysis o f B-galactosidase activity in cellular lysates was performed using the B-galactosidase Enzyme Assay System (Promega), following the manufacturer’s instructions. B-galactosidase catalyzes the hydrolysis o f o-nitrophenyl-g-o- galactopyranoside (ONPG) to produce o-nitrophenol, which can be measured by absorbance at 420nm. Briefly, cellular lysate containing a pre-determined amount o f soluble protein (approx. 50pg), was mixed with an equal volume o f 2x assay buffer (Promega: 120mM Na2HP0 4, 80mM NaH2P0 4, 2mM MgCL, lOOmM B-

mercaptoethanol, 1.33mg/ml ONPG), vortexed briefly and incubated at 37°C for 30 mins.-3hrs.. The reaction was terminated by addition o f N a2C0] to 0.625M. Absorbance was measured at 420nm and absolute B-galactosidase activity calculated by comparison to standards assayed in parallel.

2.2.16.2 Qualitative assay o f B-galactosidase activity by in

situ

A cytospin o f 10^ cells was prepared as described in section 2.2.10.6. Cells were fixed in 0.5% glutaraldehyde for 15mins. and carefully washed with PBS. Slides were incubated at 37°C for 4hrs. in X-gal buffer in the presence o f 1 mg/ml X-gal (filtered, added ju st prior to incubation). Cells were viewed under a Leitz microscope at 40x magnification. Cells positive for B-galactosidase activity exhibit blue staining (see Figure 4.1).

2.2.16.3 Assay o f CAT protein by enzyme-linked immunosorbant assay (ELISA)

CAT protein levels in cellular lysate were determined by ELISA using the CAT-ELISA kit (Boehringer Mannheim), following the manufacturer’s instructions. Cells were lysed by freeze/ thaw lysis or by active lysis (section 2.2.13). Individual CAT-antibody-coated microtitre plate (MTP) modules were rehydrated in sample buffer (Boehringer Mannheim: PBS containing blocking agents, lyophilisate dissolved in 100ml ddH20) for 5mins. at room temperature. Cellular extract containing 50pg soluble protein in 200pl sample buffer was loaded into MTP modules. The plate was covered in foil and incubated at 37°C for Ihr., or for 2hrs. if samples were o f low CAT activity. The MTP modules were emptied and washed four times with 250pl Ix wash buffer (Boehringer Mannheim: phosphate buffer containing NaCl and Tween®20, diluted to Ix with ddH20), each time leaving the wash buffer in the MTP modules for 30 seconds before discarding. Anti-CAT-digoxigenin (DIG) antibody (2pg/ml, dilution made in sample buffer) in a volume o f 200pl was added to each MTP module. The MTP was covered in foil and incubated at 37°C for 1 hour. The MTP modules were emptied and washed four times with 250pl Ix wash buffer, each time leaving the wash buffer in the MTP modules for 30 seconds before discarding. Anti-DIG-peroxidase (POD) antibody (150mU/ml, dilution made in sample buffer) in a volume o f 200pl was added to each MTP module. The MTP was covered in foil and incubated at 37°C for Ihr.. The MTP modules were emptied, washed four times with 250pl Ix wash buffer, each time leaving the wash buffer in the MTP modules for 30 seconds before discarding. POD substrate ABTS® (Boehringer Mannheim: 200pl, made up from lyophilisate dissolved in sodium perborate, citric acid/ phosphate substrate buffer) was added to each MTP module and the MTP was incubated at room temperature for 30-60mins.. Positive samples are green in colour. The absorbance o f the samples at 405nm was measured on an Anthos 2010 plate reader (Anthos Labtec Instruments, Salsburg). A reference wavelength o f 490nm was used to measure background substrate absorbance. A range o f CAT enzyme standards (O.OO-l.OOpg/pl, diluted from lOOng/ml stock (lyophilisate dissolved in ddH20) in sample buffer) were assayed in parallel. Absolute CAT protein levels were calculated by comparison with absorbance values o f CAT standards (see Figure 4.2).

2.2.16.4 Assay o f CAT activity by thin layer chromatography (TLC)

CAT enzymatic activity was assayed by TLC. CAT enzyme activity is determined in cellular lysate by the extent o f acétylation o f [^ ^C]-chloramphenicol substrate in the presence o f acetyl cofactor A (CoA). Substrate and differentially-acetylated reaction products are separated by TLC, and visualised by autoradiography. Briefly, cells were lysed by the freeze/ thaw method (section 2.2.13.1). Cellular extract containing a defined amount o f protein (approx. 50-200pg) in lOpl 0.25M Tris-HCl pH 7.8 was incubated at 60°C for lOmins. in order to inactivate endogenous deacetylase activity. Deactivated lysate was chilled on ice and centrifuged at 13000rpm for 30 seconds. Extract was incubated at 37°C for 20hrs. in the presence o f 0.55mM acetyl CoA (sodium salt) and 0.0575pCi [^"^C]-chloramphenicol (Amersham Life Science, 25|iCi/m l) in a total volume o f 168.7pl. A positive control reaction containing lU E.coli

CAT was performed in parallel. Extract was centrifuged at SOOOrpm for 30 seconds. One ml ethyl acetate (BDH-Merck, ‘Analar’ grade) was added in order to terminate the acétylation reaction. Extract was vortexed for 30 seconds and centrifuged at 13 OOOrpm for 2mins. in order to achieve phase separation. One ml o f the upper, organic phase was transferred to a fresh 1.5ml tube and dried under vacuum for 45mins.. The residue was resuspended in 20pl ethyl acetate and loaded into position on a plastic silica gel TLC plate (BDH-Merck) in lOpl aliquots. Samples were loaded at a position 1.7cm from the bottom o f a 10cm x 20cm silica plate at 1.5cm horizontal intervals. The plate was placed vertically into a chromatography tank (Orme Technologies, Fisher Scientific UK, Middleton, Manchester, U.K.) that had been pre-equilibrated for 1 hour with 200ml 90% chloroform, 10% methanol. Chromatography proceeded for 13mins.. The silica plate was removed, air-dried and exposed to Hyperfilm 6-max (Amersham Life Science) (section 2.2.9).