This experiment was aimed to learn how to do cell fraction or Sucrose centrifugation, observe cell organelles by using specific dyes and calculate DNA concentration.
Differential centrifugation is common method to separate organelles of cell from cytosol and membrane. The separation of a specific organelle is based on differences of segmentation rate of organelles. This rate is related with size, shape and density of the organelle. 1,2 and 3
Sucrose gradient centrifugation is used to achieve purer cellular fractionation mostly. Sucrose gradient forms layers to separate fractions of each other and it helps clearer difference between layers. Sucrose gradient centrifugation is used often for cellular fractionation.1,2 and 3
Nucleus is a cellular organelle being enclosed and having genetic material of cell in eukaryotic cells. Nucleus is a control part of cell and does this mission by gene expression. This gene expression regulates cell organization. 3
Mitochondrion is a kind of energy convertor for all living eukaryotic cells. Mitochondria are thought of they evolved from bacteria because of their own membrane, DNA, RNA, ribosomes and this antique symbiotic relationship formed an organelle for first eukaryotic cells. Eukaryotic cells generate many of ATP by using reactions in mitochondria. These reactions are formed between membranes of mitochondria. Enzymes and transporter on membranes served this production.3, 4
Spectrophotometry is a measurement technique for quantitate by reflection of specific wavelength of a material. Each material has specific electron order. Spectrophotometry sends a beam having a certain wavelength and this beam induce electron to higher level by giving energy.5,6
Two dyes were used for this experiment, aceto-orcien and Janus green B. Aceto-orcien is dye for staining chromatins and we can see nucleus by this method. Janus Green B stain mitochondria and it can show mitochondria activity. When oxygen is present, dyed mitochondria looks pink; when oxygen is absent, mitochondria looks blue.5,6
Material and methods:
Firstly, the liver piece was cut to small portions. Some of the small portions were placed into mortal to smash by petrel with 10 ml of 0.25M sucrose solution until solids were disappeared. The sample was transformed into a falcon tubes to perform in centrifuge for 5 minutes at 800 rpm. 5 ml of the supernatant was separated from tube and kept in Tube H (Homogenate). The residual of homogenate was centrifuged again at 5000 rpm for 15 min. After centrifuging, we had a nuclear pellet and then 5 ml of 0.25M sucrose solution was added into the tube for suspending. The nuclear fraction was separated in Tube N by labelling. Centrifuge process was used again at 10000 rpm for 30 min to achieve. After resuspending mitochondrial pellet with 0.25M sucrose, we kept the solution and we labelled as Tube M for mitochondrial fraction. All samples (Tube H, N, and M) were placed into fridge (at about 200 C). This cold condition helps for stabilizing liver enzymes by inactiving.
1 ml 0,25 M sucrose was used for filling to 15 eppendorf tubes. 5 tubes were labelled as H1, H2, H3, H4 and H5 for homogenate fractions. N1, N2, N3, N4 and N5 terms as labels are used for nuclear fraction. Same process was done for mitochondrial fractions. Initial eppendorf tubes were used as stock solution containing 1.25 ml and for tubes H, N and M. 1.25 ml of Tube H was put into Tube H2 and then mixed by using micropipette gently. Same process was used from 1.25 ml of Tube H2 to Tube H3. This processes repeated for all Tube Hs and Ns and Ms. It is called as serial dilution. 250 µl samples were taken from each tube to place into plates for spectrophotometer analysis. Next step, samples were performed under light microscopes by using dyes. Small amount of Tube N was taken and Aceto orcein was added onto slides and then cover slide was closed gently against bubbles for observation of nuclear fraction. Tube M was performed under microscope by using Janus Green B.
⦁ The value of A260= [Each value from spectrophotometer]-[Average of blank] :
Average of blank= (3,575+3,573+3,380+3,463+3,475+3,458)/6=3,487
Tubes H 0.25M sucrose Previous tube Final volume Final dilution factor [Each value from spectrophotometer]-[Average of blank] 260nm absorption:
1 1.25 ml Stock Solution No result No result
2 1 ml 0.25 ml 1.25 ml 1/5 No result No result
3 1 ml 0.25 ml 1.25 ml 1/25 3,637-3,487 0,211
4 1 ml 0.25 ml 1.25 ml 1/125 3,549-3,487 0,15
5 1 ml 0.25 ml 1.25 ml 1/625 3,549-3,487 0,062
6 1 ml 0.25 ml 1.25 ml 1/3125 3,478-3,487 -0,009
Table 1.1- Values of absorption for tubes H
Tubes N 0.25M sucrose Previous tube Final volume Final dilution factor [Each value from spectrophotometer]-[Average of blank] 260nm absorption:
1 1.25 ml Stock Solution 3,466-3,487 -0,021
2 1 ml 0.25 ml 1.25 ml 1/5 3,403-3,487 -0,084
3 1 ml 0.25 ml 1.25 ml 1/25 3,501-3,487 0,014
4 1 ml 0.25 ml 1.25 ml 1/125 3,529-3,487 0,042
5 1 ml 0.25 ml 1.25 ml 1/625 3,543-3,487 0,056
6 1 ml 0.25 ml 1.25 ml 1/3125 3,629-3,487 0,142
Table 1.2- Values of absorption for tubes N
Tubes M 0.25M sucrose Previous tube Final volume Final dilution factor [Each value from spectrophotometer]-[Average of blank] 260nm absorption:
1 1.25 ml Stock Solution 3,357-3,487 -0,13
2 1 ml 0.25 ml 1.25 ml 1/5 3,476-3,487 -0,011
3 1 ml 0.25 ml 1.25 ml 1/25 3,609-3,487 0,122
4 1 ml 0.25 ml 1.25 ml 1/125 3,571-3,487 0,084
5 1 ml 0.25 ml 1.25 ml 1/625 3,582-3,487 0,095
6 1 ml 0.25 ml 1.25 ml 1/3125 3,573-3,487 0,086
Table 1.3-Values of absorption for tubes M
⦁ DNA concentration (µg/ml) =A260*50µg/ml*dilution factor
Tubes H DNA concentration
1 No result
2 No result
Table 2.1-DNA concentrations of homogenate fractions
Tubes N DNA concentration
Tubes M DNA concentration
Cellular fraction was done according to procedure. In this part, as a difference, we separate last solution and kept it too. Because mitochondria can cause un separated complex and it can cause a wrong observation under microscope. We kept some M fraction for next part of experiment for any issue.
Fractions were performed according to how they have DNA by using spectrophotometer with 260 nm wavelengths. Then we calculated using DNA concentration formula. However we needed 260 nm absorption values. We used blank samples to calculate absorption better. We took average and subtracted from the value readied to find real absorption for DNA. When some values were investigated, these can be unexpected. Because these values can’t be expected negative. We can think of samples have been contaminated. Some contaminant could cause a change of absorption values. This is best explanation for negative results. Another reason can be device. The device has readied measurements. (Table 1.1, 1.2 and 1.3)
According to the results of the DNA concentration after calculations, the concentration increases without negative values coming from first tables. If we investigate right values, serial dilution can provide denser DNA concentration.
Finally dyes were used for last observation. Aceto-orcien couldn’t give a clear image, because of pellets and some contaminants prevented (Figure 3.1 and 3.2). We couldn’t achieve taking good observations under 10x and 40x. At least Janus Green B gave a clear image for mitochondria (Figure 3.3). Blue colour could be seen easily. This blue colour says to us that mitochondria are dead. At oxygen absent, Janus green B gives blue colour.
Chapter 3: Cell Fractionation – Introduction, Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College
De Duve, C. Exploring cells with a centrifuge. Nobel Lecture. 1974
Lodish, H; Berk A; Matsudaira P; Kaiser CA; Krieger M; Scott MP; Zipursky SL; Darnell J. (2004). Molecular Cell Biology (5th ed.). New York: WH Freeman.
Henze K, Martin W; Martin, William (2003). “Evolutionary biology: essence of mitochondria”. Nature. 426: 127-8.
Allen, D., Cooksey, C., & Tsai, B. (2010, October 5). Spectrophotometry. Retrieved (from http://www.nist.gov/pml/div685/grp03/spectrophotometry.cfm)
McBride HM, Neuspiel M, Wasiak S (2006). “Mitochondria: more than just a powerhouse”. Curr. Biol. 16 . (doi:10.1016/j.cub.2006.06.054).