COURSE CODE: BIOL 3361 TITLE OF LAB: Subcellular fractionation of liver AIM: To determine the effect of enzyme concentration

by varying the amount of invertase enzyme in Nelsons assay determine the relationship between them by construction of a graph To determine the effect of incubation time by varying the incubation time in 0,1,2,4,8,10,12,15 and 20 minute intervals in Nelsons assay determine the relationship between them by construction of a graph To determine the effect of substrate concentration on invertase activity (the rate of sucrose hydrolysis) by varying the concentration of sucrose in the presence and absence of 0.25 mL 4M urea determine the relationship between them by construction of a graph To determine the molecular weights of the components of the fractions 1 to 4 via polyacrylamide electrophoresis and subsequent comparison with a molecular larder.

THREOY: PROCEDURE:
Isolation medium : 0.25M containing 0.1mM EDTA, 1mM 2-SH-ethanol and 5mM Tris-HCL buffer pH 7.4 (cold) Standard protein solution (200g/mL BSA) Reagent A 2% Na2CO3 in 0.1M NaOH Reagent B 0.5% CuSO4.5H2O in 1% sodium citrate Reagent C Mix just before using 100mL of A and 2mL of B Folin Ciocalteus Reagent Diluted 1:1 with H2O just before using 2.5% ammonium molybdate in 2.5M Sulphuric Acid Ascorbic Acid 1g/10mL (freshly prepared) Reagent D (Prepared just before using) water : ascorbic acid : acid-molybdate in the ratio 2 : 1 : 2 Phosphate standard 2.18mg of KH2PO4 per 100mL. Maleate buffer 0.1M, pH 6.5 EDTA 10mM (in buffer pH 6.5) Glucose-6-phosphate 0.2M (in buffer, pH 6.5) 10% Trichloroacetic acid (cold) Acetate buffer 0.2M, pH 4.7 8mM p-nitrophenol phosphate (freshly prepared) Tris-HCl 1M pH 9.0 containing 1M Na2CO3 and 0.4M K2HPO4 100M p-nitrophenol standard

Triton X-100, 10% (w/v) Sodium phosphate buffer 0.1M, pH 7.4 Sodium -ketoglutarate 0.15M in buffer, adjusted to pH 7.4 EDTA 30mM in buffer, pH 7.4 Ammonium acetate 0.75M in buffer, pH 7.4 Triton X-100 10% (w/v) in buffer, pH 7.4 NADH 2.5mg/mL in buffer, pH 7.4 (freshly prepared) Sodium phosphate buffer 0.01M, pH 7.4 Sodium pyruvate 0.021M in buffer pH 7.4 (freshly prepared) NADH 3.5mM in buffer pH 7.4 (freshly prepared) Sodium phosphate buffer 0.01M, pH 7.4 Sodium pyruvate 0.021M in buffer pH 7.4 (freshly prepared) NADH 3.5mM in buffer pH 7.4 (freshly prepared) 0.35% Na2S2O4 - freshly prepared in ice-cold distilled water. This solution was used with 90 minutes of preparation. 0.1% NT freshly prepared 0.02M sodium succinate pH 7.4 0.5M phosphate buffer

Preparation of Fractions A starved rat was killed and exsanguinated .The liver was quickly removed and lightly blotted on filter paper. Approximately 8 10g of the liver was weighed, rinse twice with a small volume of cold sucrose to remove blood and placed in a beaker, on ice. It was then cut into small pieces and enough sucrose to make a 20% homogenate (10g in 50mL). The homogenate was then transferred to a homogenizing tube and homogenized with no more than 6 passes of the pestle.An equal volume of medium was added to yield a 10% homogenate and poured into two centrifuge tubes .The centrifuge scheme below was then followed.6mL of the nuclei free homogenate was saved.

Each of the pellets were re-suspended in 10mL of cold sucrose via homogenization at a very low speed . All fractions were kept on ice during the procedure. The exact volume of each fraction was measured and recorded. Half of each fraction was placed into labeled capped containers and stored for session 3 in the deep freeze. 7 tubes were prepared for the protein determination calibration curve as follows. 1.00 mL of water was placed in the test tube labeled 1. 0.10 mL Standard protein solution (200g/mL BSA) and 0.90 mL of water was placed in the test tube labeled 2. 0.30 mL Standard protein solution (200g/mL BSA) and 0.70 mL of water was placed in the test tube labeled 3. 0.50 mL Standard protein solution (200g/mL BSA) and 0.50mL of water was placed in the test tube labeled 4. 0.50 mL Standard protein solution (200g/mL BSA) and 0.50 mL of water was placed in the test tube labeled 5. 0.70 mL Standard protein solution (200g/mL BSA) and 0.30 mL of water was placed in the test tube labeled 6. 1.00 mL Standard protein solution (200g/mL BSA) was placed in the test tube labeled 7.

A x100 and x200 dilution of each of the 5 fractions were made with distilled water. 1.00 mL x100 and x200 dilution of homogenate were placed in separate test tube labeled H1 and H2 respectively.

1.00 mL x100 and x200 dilution of mitochondria were placed in separate test tube labeled M1 and M2 respectively. 1.00 mL x100 and x200 dilution of lysosomes were placed in separate test tube labeled L1 and L2 respectively. 1.00 mL x100 and x200 dilution of microsomes were placed in separate test tube labeled Micro1 and Micro2 respectively. 1.00 mL x100 and x200 dilution of soluble fraction were placed in separate test tube labeled SF 1 and SF 2 respectively. 1.00 mL of Lowry reagent C was added to each tube. The tubes were left to stand at room temperature of 10 minutes. 0.5 mL Folin-Ciocalteus reagent (diluted) was added to each tube which were vortexed. The tubes were left to stand for 30 minutes at room temperature ,after which the absorbance for each was measured at 750 nm and recorded. 7 tubes were prepared for the inorganic phosphrous calibration curve as follows. The tubes were pre-washed with acid before use. 0.00 mL standard phosphate solution and 5.00 mL of water was placed in the test tube labeled 1. 0.50 mL standard phosphate solution and 0.50 mL of water was placed in the test tube labeled 2. 1.00 mL standard phosphate solution and 4.00 mL of water was placed in the test tube labeled 3. 2.00 mL standard phosphate solution and 3.00 mL of water was placed in the test tube labeled 4. 2.00 mL standard phosphate solution and 3.00 mL of water was placed in the test tube labeled 5. 3.00 mL standard phosphate solution and 2.00 mL of water was placed in the test tube labeled 6. 4.00 mL standard phosphate solution and 1.00 mL of water was placed in the test tube labeled 7. 4.00 mL of Reagent D was added to each tube and the tubes were then mixed via vortexing. The tubes were then immediately placed in a water bath at 60 C for 10 minutes. After the tubes cooled down,the absorbance was read and recorded at 650nm.

15 tubes were prepared for the assay of Glucose-6-phosphatase as follows. The small tubes were pre-washed with acid before use. 0.6mL Buffer, 0.1mL EDTA and 0.2mL glucose-6-phosphate were placed in each tube,which were left to Equilibrate at 35 C for 5minutes. Five of the tubes were zero time tubes labeled H0, M0, L0, Micro0 and SF0. 1 mL of 10% TCA was added to each tube before adding the corresponding 0.1 mL of fractions and incubated at 35 C for exactly 10 minutes. The other ten tubes will serve as duplicates labeled H1,H2, M1,M2, L1,L1, Micro1,Micro2,SF1 and SF2. The reaction was started by adding 0.1mL of the respective fractions to the appropriate tubes and incubated at 35 C for exactly 10minutes . The reaction in the tubes were terminated by adding 1mL of the cold 10% TCA solution at the end of 10 minutes. The tubes were then Centrifuged at 3000rpm for 10 minutes in the bench centrifuge and 1mL of the supernatant was removed for phosphate determination as described above. 7 tubes were prepared for the Assay of Acid Phosphatase calibration curve as follows. 1.00 mL of water was placed in the test tube labeled 1. 0.20 mL 100M p-nitrophenol and 0.80 mL of water was placed in the test tube labeled 2.

0.40 mL 100M p-nitrophenol and 0.60 mL of water was placed in the test tube labeled 3. 0.40 mL 100M p-nitrophenol and 0.60 mL of water was placed in the test tube labeled 4. 0.60 mL 100M p-nitrophenol and 0.40 mL of water was placed in the test tube labeled 5. 0.80 mL 100M p-nitrophenol and 0.20 mL of water was placed in the test tube labeled 6. 1.00 mL 100M p-nitrophenol was placed in the test tube labeled 7. 1.2mL of acetate buffer and 2mL of alkaline Tris buffer was added to each tube.each tube was then vortexed and the absorbence read at 405nm against the blank. 10 tubes were prepared for the Assay of Acid Phosphatase without detergent as follows. Five zero-time control where labeled H,M,L,Micro and SF. 1.2mL of acetate buffer, 0.5mL of substrate solution, 0.1mL of water and 2mL of alkaline Tris buffer was added to each tube. Each tube was vortexed. Five other normal reaction tubes were prepared labeled H1,M1,L1,Micro1 and SF1.1.2mL of acetate buffer, 0.5mL of substrate solution and 0.1mL of water was added to each tube. each tube was vortexed.all the tubes were incubated for 5 minutes at 35 C. 1 in 10 dilutions for all the fractions were prepared for each fraction. the reactions in each tube were started by adding 0.2mL of the corresponding diluted fraction sample,the tubes were vortexed and incubated for 10 minutes. The reactions in the normal reaction tubes were Terminated by adding 2mL of alkaline Tris buffer to each tube and vortexing. tThe tubes were allowed to cool down, after which the absorbance of the solution was read at 405nm and recorded. Assay of lysosomal fraction with the detergent Triton X-100 was done with two tubes. 1.2mL of acetate buffer, 0.5mL of substrate solution, 0.1mL of 10% Triton X-100 and 2mL of alkaline Tris buffer was added to the zero time tube labeled Lo,and the tube vortexed. 1.2mL of acetate buffer, 0.5mL of substrate solution and 0.1mL of 10% Triton X-100 was added to the normal tube labeled L1,and the tube was vortexed.The tubes were incubated for 5 minutes at 35 C.

The reactions in each tube were started by adding 0.2mL of the 1 in 10 diluted lysosomal fraction sample. The tubes were vortexed and incubated for 10 minutes. The reaction in the normal reaction tube was Terminated by adding 2mL of alkaline Tris buffer and vortexing. The tubes were allowed to cool down, after which the absorbance of the solution was read at 405nm and recorded. The Determination of the Marker Enzymes used 10 tubes as follows. A 1 in 10 dilution of each fraction was prepared . 2.1 mL Phosphate buffer ,0.1 mL NADH,0.2 mL Ammonium acetate,0.2 mL EDTA and 0.1 mL Triton X-100 were added to each of the 10 tubes. The tubes were labeled H,M,L,Micro,SF, H1,M1,L1,Micro1 and SF1.each fraction had 2 tubes. 0.2 mL of the respective diluted fraction was placed into the tubes which were then vortexed. 0.1mL of the substrate -ketoglutarate was added to tube H ,which was then vortexed, to start the reaction. The solution was Transfer immediately to a cuvette and the change in absorbance at 30-second intervals over a 4-minute period was read and recorded .this was repeated for each of the other 9 tubes. The Determination of the Lactate Dehydrogenase Marker Enzymes used 10 tubes(each reaction was done in duplicate) as follows.A 1 in 50 dilution of each fraction was prepared . 2.6 mL Phosphate buffer and 0.1 mL NADH were placed into each of the 10 tubes. The tubes were vortexed and allowed to equilibrate at 37 C for 10 minutes.At the spectrophotometer 0.1mL of the substrate sodium pyruvate solution was added to the tube with the homogenate

fraction to (to start the reaction) and the tube vortexed. Immediately after, the solution was transfered to a cuvette and the change in absorbance was read and recorded at 30-second intervals over a period of 4 minutes at 340nm. The Calibration Curve for Determination of the subcellular location of Succinate dehydrogenase used 10 tubes as follows. the 0.1% NT was diluted to 0.05% to make 5ml. 1.00 mL of water was placed in the test tube labeled 1. 0.10 mL 0.05% NT and 0.90 mL of water was placed in the test tube labeled 2. 0.20 mL 0.05% NT and 0.80 mL of water was placed in the test tube labeled 3. 0.20 mL 0.05% NT and 0.80 mL of water was placed in the test tube labeled 4. 0.30 mL 0.05% NT and 0.70 mL of water was placed in the test tube labeled 5. 0.40 mL 0.05% NT and 0.60 mL of water was placed in the test tube labeled 6. 0.60 mL 0.05% NT and 0.40 mL of water was placed in the test tube labeled 7. 0.60 mL 0.05% NT and 0.40mL of water was placed in the test tube labeled 8. 0.80 mL 0.05% NT and 0.20 mL of water was placed in the test tube labeled 7. 1.00 mL 0.05% NT was placed in the test tube labeled 8.

2 mL of cold Na2S2O4 was added to each tube, which were then vortexed. 7mL of acetone was added to each tube to dissolve the coloured formazan and vortexed again. The tubes were allowed stand for 10 minutes after which the absorbance at 540nm was measured and recorded.

Assayed of the fractions required 15 tubes.An enzyme blank and a substrate blank were prepared for each fraction. Each fraction was prepared in duplicate as follows: 0.5 mL of buffer,1.0mL succinate,0.05 mL 0.1% NT and 0.5 mL distilled water.the tubes were equlibriated at 35oC for five minutes and then 0.3mL of the undiluted sample was added to the substrate blank and the tubes incubated for 20 minutes. 7mL of acetone was added to each tube to dissolve the coloured formazan and vortexed again. The tubes were allowed stand for 10 minutes after which the absorbance at 540nm was measured and recorded.

Throughout this procedure ,after addition of a reagent to the tubes,vortexing was done.