Techniques_of_Cell_Disruption

Techniques of cell disruption Abstract: The aim of this experiment was to find the best type of cell disruption technique. We used 5 different techniques against each other. They were: freeze-thaw disintegration, ultrasonic disintegration, grinding with washed sand, disruption using a waring blender and disruption using a potter homogeniser. We tested them on both yeast cells (Saccharomyces Cerevisiae) and lettuce (Lactuca Sativa), the outcome of the test was to see which technique was was able to liberate the most amount of protein from the cells. Therefore the higher the protein content at the end of the experiment the better then technique is at breaking the cell wall to release the protein. Results showed that for yeast ultrasonic disintegration gave the highest protein content with 0.050 and the lowest was free-thaw which gave the result of 0.000. For lettuce disruption using a potter homogeniser gave the highest result of 0.218 and again the lowest was freeze-thaw which gave 0.000. The results were expected as lettuce has larger cells than that of yeast so the disruption methods that are designed for the lager cells e.g. disruption using a waring blender and disruption using a potter homogeniser would not give a positive result for yeast as the cells are too small to be affect by the technique. Introduction: This study was designed to demonstrate different methods of cell disruption and their efficiency using two types of biological material, yeast cells and lettuce. It aims to determine the efficiency of disruption by determining the protein released per gram-wet weight of starting material. Five different methods of cell disruption were used. Each method involved suspending the material in buffer, subjecting it to the disruption technique, and centrifuging to remove debris. The success of the different methods was determined by measuring the total amount of protein released by each treatment by use of the Folin and Ciocalteu method. This method depends mostly upon a reaction with phenolic hydroxyl groups. Its reaction with proteins and protein-breakdown products is, therefore, based largely upon their tyrosine content, since this is the only amino acid containing such groups, although the tryptophan and cysteine moieties also react to some extent. In the presence of other phenolic compounds spurious results may be obtained, e.g. in serum under certain pathological or toxic conditions. Globulins are reported to contain 4-13% less tyrosine than albumins, but, in general, mixtures of proteins such as in tissue fluids and tissue extracts can be estimated satisfactorily by this method, which is rapid and sensitive and is therefore attractive for analysis of material such as spinal fluid, which is available only in very limited quantities. Method: 2.5 g wet weight sample of yeast or lettuce were placed in 0.01 M phosphate buffer pH 7.4 and subject to a variety of disruption methods. All procedures were carried out at 0 to 5C. The lettuce preparations were bleached after centrifugation to destroy pigmentation that can interfere with the protein assay. All the preparations were adjusted to 50 ml with buffer. These methods included the following: (1) Freeze-thaw disintegration, samples were frozen and thawed five times using 24 hr cycles. (2) Ultrasonic disintegration, samples were subjected to ultrasound for 10 minutes. (3) Grinding with washed sand, samples were made up in minimal buffer and semi-frozen in a pestle and mortar taken from a deep freeze. 1-2 g of coarse washed sand was added and grinding carried out for 10 minutes. (4) Disruption using a Waring blender, samples were blended for five minutes. (5) Disruption using a Potter Homogeniser, samples were homogenised for five minutes. Protein concentrations of the samples were determined using a Folin and Ciocalteu assay. 5 ml of Folin reagent F was mixed with 1ml of sample and allowed to stand for 10 min. 0.5 ml of Folin reagent D was added and the sample was allowed to stand for 30 minutes before the optical absorbance was measured at 660 nm on a spectrophotometer, against a blank containing no sample. A calibration curve was constructed using bovine serum albumen (0-0.2 mg/ml) as a sample source of protein. The assays were performed in duplicate. Results: The result of this experiment was to be able to determine which method of cell disruption is the best; results were produced by finding out how much protein was removed from inside the cell thus telling us which is the best method of cell disruption. Therefore, the greater the protein content the better the method is at disrupting the cell wall. Cell disruption is important as it gives scientists a chance to get into cells to see if anything is abnormal. With the lettuce the most effect cell disruption technique was the potter homogeniser as it gave a protein content of 0.218 and the least effective was using the freeze-thaw method which didn’t even produce protein content. The yeast results didn’t vary much between the different techniques. Ultrasonic disintegration and grinding with washed salt were the only techniques which gave a result. However, the protein content that was produced was very low. 0.050 was the highest and that was produced by the ultrasonic disintegration, the other three techniques did not produce any protein content. Fig 2 shows the standard cure that was performed so that we could work out the protein concentrations that the yeast and lettuce produced. Table 1. Protein released from fractions. Fraction Absorbance at 660 nm (sample 1) Absorbance at 660 nm (sample 2) Mean absorbance at 660 nm Protein content (mg/ml) Y1 0.000 0.000 0.000 0 Y2 0.037 0.160 0.099 0.050 Y3 0.110 0.004 0.057 0.0280 Y4 -.124 -.087 -.106 0 Y5 -.076 -.068 -.072 0 L1 0.000 0.000 0.000 0 L2 0.093 0.061 0.077 0.038 L3 0.101 0.257 0.179 0.088 L4 0.306 0.384 0.345 0.172 L5 0.466 0.458 0.462 0.218 Fig 1. Fig 2. Discussion: The overall results show that yeast is less affected by the different techniques compared to lettuce (fig 1). The lettuce samples gave positive results where the yeast cells didn’t produce any result. Only with the freeze-thaw method did both results give a negative result. The results that were obtained I think were expected, the only unknown was just how much protein would have been collected. For example we knew that ultrasonic disintegration and grinding with washed sand would produce a positive result for both lettuce and yeast, they are both methods that are able to disrupt the cell walls of smaller cells (yeast) and slightly larger cells (lettuce). We can prove this as disruption using a potter homogeniser did not give a positive result of protein for yeast but it did for lettuce, this is because the sand is a lot smaller that the potter homogeniser and was able to disrupt the cell wall. Therefore, if we consider the waring blender we know why there is no positive protein count for yeast as the blades that are used are far too large to have any affect. Fig 1 also gives us a good idea of how each of the methods compared to each other, showing if there was a positive result or not. Yeast can be extremely difficult to disrupt because their cell walls may form capsules or cells are nearly indestructible spores. Incomplete disruption will also result in decreased yield because some of the protein in the sample remains trapped in intact cells and, therefore, is unable to affect the results. The Folin-Ciocalteu’s phenol reagent reacts primarily with tyrosine residues in the protein, which can lead to variation in the response of the assay. If there is a low amount of tyrosine then the results would be affected. From the results gathered it is hard to say which the best method for disrupting the cells is. We are trying to compare two different species with different disrupting techniques. References: Strettle, R. (2003) Unpublished Biology Skills Workbook Internet sites: http://www.nihs.go.jp/dbcb/protein.pdf