Cell Homogenization, Cell Fractionation or Isolation, and Purification of Chicken Liver Tissue through Grinding and Centrifugation

11042575 Biology Department De La Salle University

ABSTRACT This experiment aims to homogenized and fractionized chicken liver tissue to be able to quantify its protein content. Homogenization and cell fractionation are two essential steps to be able to quantify protein content of a sample. Homogenization is the disruption of the chicken liver tissue, and this is done using a blender with grinding buffer. This is followed by isolation or cell fractionation through series of centrifugation that requires 12,000rpm to be able to separate the subcellular organelles completely. Standardization is done for comparison and the protein content of the fractions is quantified. The result showed that the nuclei fraction has the highest absorbance reading of 1.049, followed by the soluble=1.356, crude=0.755, and microsome=0.017. The result showed that there is presence of contamination INTRODUCTION Scientists and researchers study subcellular components of cell to trace and see the reaction or activities present. To study these subcellular organelles, necessary steps are needed. Homogenization is the first step that disrupts the cell or the tissue. This can be done by grinding, ultrasonic vibration, osmotic shock, or enzymatic digestion (Hardin et. al, 2012). Homogenization in the study is done by grinding using a blender because this is the easy way to homogenize a chicken liver. Chicken liver tissue is used to this study because of its soft tissue that is easy to blend. If the sample is from a higher structure animal tissue like muscle, isolation of subcellular organelles might be difficult because of the high proportion of the organelles remain trapped in the cell and tissue fragments like the mitochondria. Substantial quantity of subcellular organelles can be easily and quickly obtained by using chicken liver tissue than any higher type of tissue (Caprette, 2005). Centrifugation follows the homogenization. Centrifugation (Fig.1) is the method of the isolation and purification of the subcellular organelles from the homogenized tissue. The sample is subjected to centrifugal force by spinning the sample at an utmost rapid rate in a centrifuge. In addition to this, the rate of the movement of the components of the sample, or the particles, depends on its size and density, as

well as the density and viscosity of the solution. As the centrifuge spins, sub cellular organelles separate based on their density and size. This relative size or density can be express in terms of sedimentation coefficient. This is a measure that shows how fast the organelles when subjected to a centrifugation and this are expressed in Svedberg units (S). This unit honors the Late Theodor Svedberg, a Swedish chemist that developed the ultracentrifuge (Hardin et. al.). Ultracentrifuge has a maximum of 100,000 rpm that means that it is can widely separate organelles in cell biology, biochemistry class and nano-material fields (Ultracentrifuge, u.d.).

(http://cellbiologyolm.stevegallik.org/node/74)

Figure. 1 The centrifuge and its parts used to isolate subcellular organelles with maximum of 12,000rpm. There are different types of centrifugation (Table 1). These include differential centrifugation, which is used in the experiment. This separate particles based on their density and undergoes series of centrifugation, which supernatant or the liquid suspension of the homogenate is used for the next centrifugation until specific fraction is obtained: nuclei (where nucleus can be found), microsome (fragments of endoplasmic reticulum) and soluble (proteins); density gradient centrifugation the variation of the of differential centrifugation in which sample for fractionation is placed as a thin layer on top of a gradient solute that increases the density from the top of the tube to the bottom and when centrifuged, specific places or spots will be observed and this means it separated; and equilibrium density centrifugation also known as the buoyant density like the density gradient centrifugation, however, the

solute used here is a very concentrated solute so that the range of the densities of the organelles are far enough or separated equally (Hardin et. al., 2012). Table 1. Differential centrifugation, density gradient centrifugation and equilibrium density centrifugation differs from the separation technique. Equilibrium Density Differential Centrifugation Density Gradient Centrifugation Centrifugation

(http://www.sigmaaldrich.com/technical-documents/articles/biofiles/centrifugation-separations.html)

There are Novel prize winners to cell biology on 1974 that are awarded for pioneering the work of centrifugation and subcellular fractionation. These are Albert Claude, the one that developed differential centrifugation as a procedure in isolation of subcellular organelles; George Palade, used centrifugation to study the endoplasmic reticulum and the Golgi complex which their roles in the cell such as biosynthesis, processing, and secretion are established; and Christian de Duve who also used centrifugation to study the lysosomes and peroxisomes. Lysosomes are discovered by De Duve wherein hes interest to study the effect of the insulin on carbohydrates, he accidentally used a different enzyme that has different activity that when observed in a microsope, it is not the mitochondria, it is membranebounded vesicles or lysosomes and became unique feature of animals. (Hardin et. al., 2012). This experiments objective is to quantify the protein content of the fractionated sample where the crude has the highest protein content since it is the original sample and unfractionated, next is the soluble because it contains proteins, then nuclei because it has macromolecules like DNA and RNA, and lastly, the microsome that is composed of lysosomes, peroxisomes and mitochondria through series of centrifugation or the differential centrifugation. MATERIALS AND METHOD A. Preparation of fractions

For the entire class, 50 grams of chicken liver tissue was blended with 150mL of grinding buffer. Grinding buffer was an isotonic solution that preserves the integrity of the subcellular organelles (Hardin et. al., 2012). The large chunks were broke and it was blended until it was completely liquefied. Then it was filtered using cheesecloth and squeezed the juice so that there will be no waste of materials. Then each group of the class collected four (4) microfuge tubes with 1.0mL of homogenized and filtered sample. The two (2) tubes were labeled as the CRUDE. There were 2 crude samples so that it also has a replicate that just in case missing, there was a replacement. Then the crudes were stored in an ice bucket. The two tubes left were centrifuged at 12,000 rpm for 10 minutes. And it was ensured that the tubes were placed balanced. This should be 13,000 rpm. However, the centrifuge only has 12,000 rpm as its maximum. After centrifugation, the supernatant from the 2 tubes were discarded without disturbing the pellet, the substance at the bottom of the tube. Then the pellet was resuspended by adding 1 mL of grinding buffer then centrifuged again at 12,000 rpm for 10 minutes. The supernatant was transferred into a separate clean microfuge tubes. Then the pellet was resuspended with 1 mL grinding buffer and labeled as the NUCLEI and placed in the ice bucket. The supernatant transferred to clean microfuge tubes previously were centrifuged again at 12,000 rpm for 10 minutes. The supernatant from two tubes were transferred into clean microfuge tubes and labeled as the SOLUBLE and placed in the ice bucket. The pellet left was resuspended with 1 mL of grinding at 12,000 rpm buffer for 15 minutes since the pellet was too tiny. The supernatant was discarded and the pellet was resuspended by adding 1 mL of grinding buffer then labeled MICROSOME then placed in ice bucket. Each fraction has 2 microfuge tubes. B. Preparation of Standard Curve and Fractions for Absorbance A new standard curve (Fig. 2) was made to associate the set of protein solutions of known concentrations to the unknown. Just like experiment 1, five (5) microfuge tubes were used with certain amount of albumin and another one for blank without albumin. Then instead of PBS, certain amount of grinding buffer was added to the albumin. Then Bradford reagent was added last and it was vortexed then settled for 10 minutes. The amounts were shown at Table 2 as well as the concentration of the albumin. Then the absorbance was read at 595 nm. Fractions were prepared by adding 200ul of grinding buffer to 50ul of each fraction. Then 250ul of Bradford reagent was added. It was vortexed and settled for 10 minutes. The absorbance was read at 595 nm (Table 3). RESULTS Table 2. Concentrations needed for the new standard curve and the final concentration of the albumin

Standard No.

Bradford Reagent (ul)

Grinding Buffer (ul)

Chicken Egg White Albumin (ul)

Concentration (ug/ul)

Blank 1 2 3 4 5

250 250 250 250 250 250

250 210 170 130 90 50

0 40 80 120 160 200

0 0.8 1.6 2.4 3.2 4.0

Table 3. Standard Curve Absorbance Standard No. 1 2 3 4 5 Concentration (ug/ul) 0.8 1.6 2.4 3.2 4.0 Absorbance (595nm) 0.471 0.593 0.212 0.463 0.590

Table 3 shows the absorbance read at 595 nm. There is a misleading result, however, standard no. 3-5 are used for graphing the standard showed in Figure 2.
0.7 0.6 Absorbance (595nm) 0.5 0.4 0.3 0.2 0.1 0 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 Concentration (ug/ul) R = 0.9654

Fgure 2. Standard Curve with R2=0.965 , strong positive linear relationship

Table 4. Absorbance of the Subcellular Organelles at 595nm Fraction Absorbance (595nm)

Crude Nuclei Soluble Microsome

0.755 1.049 1.356 0.017

Table 5. Original concentration of the proteins of the fractions Sample/ Fraction Blank Crude Nuclei Soluble Microsome Dilution 0 1 3 2 4 Dilution Factor 0 1 3 2 4 Abs 595 nm 0.00 0.755 1.049 1.356 0.017 [Protein] from [Protein] of the standard curve original sample 0 0 0.755 0.350 0.678 0.00425

REFERENCES: Caprette, D. 2005. Preparing Mitochondria from Rat http://www.ruf.rice.edu/~bioslabs/studies/mitochondria/mitoprep.html Ultracentrifuge. Unknown date. WX_ultra_santrifuj__NEW__.pdf Retrieved from: Liver. Retrieved from:

http://www.artlaborteknik.com/dosyalar/1_CP-