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Cryopreservation is becoming an important application due to its uses in various fields like medical science, aquaculture and also in the conservation programme. In cryopreservation cells are stabilised specifically at very low temperature that is -1960C or 77K. Cryoprotective agents are added to prevent ice formation at this much low temperature. In this research glycerol has used as a cryoprotective agent in different concentration of 10%, 15% and 20%. This cryoprotective agent may cause DNA damage. To check this DNA damage, lymphocytes are selected as a good source of DNA. Lymphocytes have become choice of selection for cryopreservation because of its different cellular function and various immunological applications. The main regulation of DNA damage checkpoints are three phosphoinositide - 3 - kinases (PIKKS) ; Ataxia Telangiectasia Mutated (ATM), ATM and Rad 3- related (ATR) and DNA dependant protein kinase (DNA-PK). ATM mostly responds to DNA double strand breaks (DSBs), while ATR is activated by Ultra Violet (UV) or stalled replication fork and also acts in a supportive role in the DSBs. To provide sufficient time for repair, cell cycle is arrested at G1, S and G2 checkpoints. At these checkpoints ATM is phosphorylated on Ser1981 which can be detected by using P-ATM antibody. By utilising Dot Blot and Western Blot techniques, this phosphorylated ATM is noticed. DNA damage has been noticed with 20% glycerol. It is concluded from the result that it will be safe to cryopreserve lymphocytes with lower than 20% of glycerol, more likely at 10% or 15% concentration.
Key words: cryopreservation- lymphocytes, cryoprotective agent- glycerol, DNA damage, ATM, ATR, ATM phosphorylation, dot blot, western blot.
A-T - Ataxia Telangiectasia,
ATM - Ataxia Telangiectasia, Mutated,
ATR - ATM-and Rad3-related,
BSA - Bovine Serum Albumin,
Cdks - Cycline dependent kinases,
DDR - DNA Damage Response,
DNA- PK - DNA-dependent Protein Kinase
DSB - Double Strand Break,
p53 - phosphorylated 53,
PBS - Phosphate Buffer Saline,
PIKKs - Phosphoinositide-3-Kinases,
PVDF - Polyvinylidene Fluoride,
Ser - Serine,
Thr - Threonine,
Cell lines in continuous culture are prone to genetic drift, finite cell lines are fated for senescence, all cell cultures are susceptible to microbial contamination, and even the best-run laboratories can experience equipment failure.Â Because an established cell line is a valuable resource and its replacement is expensive and time consuming, it is vitally important that they are frozen down and preserved for long-term storage.
Cryopreservation is storage of living organisms at ultra low temperature i.e. -1960C or 77K so that, it can be revived and restored to the same living state as before it was stored. Some living cells may be stabilised for weeks or even for years in liquid nitrogen at ultra-cold freezer temperature ( Simione, 1998 ). At low temperature, biological metabolism in living organism is weaken and finally stops. This fact allows long term cryopreservation ( Critser and Gao, 2004 ). The best method for cryopreservation of cultured cells is to store them in complete medium of liquid nitrogen in the presence of a cryoprotective agent.Â Certain small molecules are able to enter in the cells and prevent dehydration and formation of intracellular ice crystals which can lead to cell death and destroy the cell organelles during the freezing process. These small molecules are known as cryoprotective agents (Meryman, 1971). Cryopreservation of the mammalian cells became successful for the first time in 1946 when glycerol was used as a cryoprotective agent recommended by Polge et al. ( Critser and Gao, 2004 ). Since then many cryoprotective agents have been discovered and used in cryopreservation.
According to Meryman (1971), there are two different types of Cryoprotective agents: (1) Penetrating and (2) Non penetrating. Penetrating cryoprotectants enter into the cell and prevent dehydration and ice formation. Two common penetrating cryoprotectants are dimethyl sulfoxide and glycerol. Glycerol is generally used as cryoprotective agent for red blood cells while dimethyl sulfoxide (DMSO) is primarily used for cryoprotection of most other cells and tissues. Non penetrating cryoprotective agents have ability to leak solute reversibly under osmotic stress. The selection of cryoprotective agents depends on different types of cells. Glycerol has become the agent of choice because of its less toxic nature than DMSO. However glycerol also has some limitations; the major limitation is its slow movement through the membrane of cells which are permeable for it. On the other hand DMSO is more penetrating than glycerol therefore it is mostly used for larger, more complex cells such as protists.
Blood can provide classical field for cryopreservation because all different categories of blood cells ( including blood stem cells and blood corpuscles ) can be cryopreserved with the only exception of granulocytes for which a reliable protocol has not been reported yet. Frozen lymphocytes are used for various diagnostic and clinical purposes. The methods of cryopreservation depend on (1) cell concentration, (2) protective solutions used, (3) temperature-time histories and (4) storage temperature ( Sputtek, 2009 ). The major advantage of extending the shelf life of lymphocytes from weeks to years is three - fold. Lymphocytes are useful for various in-vitro immunological studies. The purpose of using glycerol is to improve the liquid storage of lymphocytes. (.........). Special cells like lymphocytes help body to fight against infection. Lymphocytes can travel to any part of the body. Mainly 3 types of lymphocytes are present in the body : (i) B lymphocytes : They originate from the bone marrow and remain in the bone marrow until they become mature. B lymphocytes recognise the bacteria and viruses. (ii) T lymphocytes : T cells move to thymus to grow fully. T cells start attacking virus and bacteria. (iii) Natural Killer cell : they kill abnormal cells, bacteria, cells infected with viruses and cancer cells ( The Guardian, 2010 ).
These days' clinical diagnosis and treatment of diseases become more and more advanced and with that, demand of cells and organ transplantation to cure acquired disease and genetic defect also increases. Besides these, more and more types of living cells, tissues, organs and engineered tissues are urgently required to be cryopreserved. Cryopreservation is becoming an important application in the field of medical science and also in conservation programmes because of its long storage capacity for biological samples at cryogenic temperature.
Cryopreservation is widely used in clinical medicine, agriculture, aquaculture and in other life science fields due to its capacity to store the cell lines at very low temperature which makes this technique more useful than other cell storage techniques. However it carries some disadvantages that it causes cytoplasmic damage which limits its uses. Furthermore, some research studies indicate its involvement in genetic DNA damage but it has not been proved ( Kopeika, et al. 2004 ). Genetic integrity and cell viability are very important for the existence and functioning of all the organisms ( R & D systems, 2003 ). In cryopreservation of lymphocytes, mostly DNA strands break and bases altered in the form of oxidized purines, oxidized pyrimidines and misincorporated uracil ( Duthie, 2002 ). According to Martin (2004), many changes like apoptosis, are induced in sperm cells by cryopreservation. When human DNA battered by endogenous cellular metabolites and exogenous damaging agents, the normal cell cycle is arrested by its checkpoints and DNA will be repaired by sequential event. The main regulators of checkpoint pathway in human DNA damage are ATM, ATR and DNA-PK, although the initial activation of these regulators is not fully understood ( Fan, et al., 2007 ).
2.1 ATM and ATR protein kinase
ATM, ATR and DNA-PK are serine/Threonine kinases of the PIKK family. In human homozygous ATM deficiency cause Ataxia - Telangiectasia (A-T) which is a radiosensitive and genome instable disorder. Patients with this disease show chromosomal instability and defects in the G1/s, G2/M and S-phase checkpoints. A-T is a type of autosomal recessive. It has frequency of 1:40,000 - 1:100,000. ATM binds with the DNA at its ends. It is easily recognised by atomic-force microscopy. ATM and ATR are very large proteins with molecular weight 350KDa. A modified model of the initiation of DNA damage signalling suggests that, PIKKs which correlates with Ku like or other lesion binding proteins, act as DNA damage sensors. ( Daniel, 2001 )
2.2 Activation of ATM and ATR
Key regulators of DNA damage signal transduction are ATM and ATR protein kinases. ATM respond to double strand breaks of DNA while ATR responds to almost all types of DNA damage and also respond to stalling of replisomes. ATM and ATR become activated when they interact with the damaged sites of DNA and phosphorylate multiple target proteins at Ser-Gln or Thr-Gln. Appropriate mechanism of rapid translocation of both of these kinases are not yet known, and other proteins associated with these sites can directly get phosphorylated by these kinases. Other mediator proteins are required by ATM and ATR while doing phosphorylation of downstream targets. These proteins include BRCA1 breast and ovarian cancer susceptibility gene product. An important regulator of genome stability is 53BP1 which is found in the two-hybrid screen with p53, which protects cells against double strand breaks.
Cells are provided by DNA repair mechanism and also cell cycle checkpoints in DNA Damage Response (DDR) which acts as biological barrier. Both endogenous and exogenous sources of DNA damage are controlled by main signalling molecules like ATM, ATR, Chk1 and Chk2. Different kinds of exogenous factors can affect DNA and act as DNA damaging factors such as UV light, IR and a large number of organic and inorganic chemical substances, these potentially include cryoprotective agents. Genetic damage may include simple DNA mutation, DNA single and double strand breaks or more complex changes. Cryoprotective agents mostly affect on DNA double strands and cause double strand break. The cell cycle can be arrested by DNA damage checkpoints until damage is repaired. Apart from regulation of DNA damage, DDR has several other functions also. It also takes part in regulation of telomere length, cell cycle arrest, activation of cell cycle death by apoptosis and also triggers some critical transcriptional programs. Moreover it can also be shown that when particular gene is knocked out which cause embryonic lethality in mice, it is regulated by some checkpoint. ATM and ATR can work as both sensor and transducer. Moreover BRCA1 also have many functions. Not only p53 and Chk2, BRCA1 is also a target for ATM. Therefore it is also phosphorylated and activated by ATM. BRCA1 make complex with many other proteins and performs an important role in DNA damage response. Apart from role in DDR, it also takes part in regulation of oxidative stress by combining with antioxidative enzyme systems.
DNA double strand breaks are detected by ATM and repaired has been done by homologous recombination. Multiple cascade pathways are evoked in response to DNA damage. Checkpoint kinases Chk1 and Chk2 are phosphorylated and activated by ATM which in turn phosphorylates CDK CD25. DDR is a fundamental part of senescence. ATM, ATR and possible DNA-Pk are activated by DDR. Microscopic foci are formed by phosphorylation of ATM/ATR target proteins near the site of damage which consist of large accumulation of proteins such as 53BP1, gamma-H2AX and phosphorylated ATM. Chk1, Chk2 and p53 are other regulatory proteins which are also activated and found throughout the nucleus. DDR proteins serve to sense the break, amplify the DNA damage, signal and formulate a cellular response. (suchismita, 2003 )
Temporary arrest of cell cycle by cell cycle checkpoint is one type of cellular response to DNA damage, variety of DNA damaging agents and stalled replication forks activate these response pathways. ATM and ATR organise the damage response sometimes together and sometimes separately. Whereas DSBs is mostly indicated by ATM while ATR also respond to UV damage and replication arrest. ATR also acts as a supportive in DSBs response. ATR has a sequence homology with ATM and Rad3. Because of sequence homology ATR has a quite similarity in function with ATM and also overlap substrate specificity. Though, ATM acts in the early stage of DNA damage while ATR comes in a later stage. In general ATR bound with Chk1 and Chk2 is become more likely target for ATM. Information for different behaviour of ATM and ATR is provided by ATM and ATR null mice model. ATM null mice show phenotypic characterization like growth retardation and infertility. On the other hand death occurs in ATR null mice because of mitotic catastrophe. In conclusion, ATR is essential for life as it participate in monitoring DNA replication. ( Angela, 2010 )
Negative effects like increased sensitivity of DNA damaging agents and defective activation of cell cycle checkpoints are increased in the ATR deficient or in the inactive ATR cells. Whereas over expression of active ATR in cells produce defect of S-phase checkpoint in A-T cells. ( Shiloha, 2001 )
Fig 1 : In vertebrate, different signals of DNA damage draw out a branch composition, ATM-Chk2 and ATR-Chk1 which is indicated by solid lines in the figure. On the other hand genetic studies show that both kinases have cross link which is shown by doted lines in the figure.
2.3 Two kinases many targets
By studying the cell cycle checkpoints at G1/S and G2/M, information can be got for ATM and ATR.
Fig 2 : regulation of cell cycle division at different checkpoints and its control by cyclines and Cdks
The G1/S and G2/M checkpoints: G1 checkpoint is best understood mammalian checkpoint which promotes replication of damaged DNA (yang, 2003). Activated and accumulated p53 protein mediates G1/S checkpoint. By this way p21 gene is activated which is an inhibitor of the cell cycle machinery. Many post-translational modification are occurred which mediate this process after p53 activation and accumulation. These modifications are mostly ATM dependent in the initial, rapid phase of DSBs response, for example Ser15. However some studies show that phosphorylation of Ser20 residue is important for stabilization of p53 instead of Ser15. Because of this modification association of p53 Mdm2 is inhibitor of p53 and act as a mediator in its degradation. ATM does not target Ser20 directly but the ATM dependent stabilization of p53 may cause the activation of Ser20.
The S-phase checkpoint is the least understood mammalian repair pathway where progression of cell cycle is monitored and the DNA synthesis would be decreased which follow DNA damage. It is experimentally proved that S-phase checkpoint gets activated by IR damage via two parallel branches and both branches are controlled by ATM. The role of ATR in S-phase checkpoint is still unclear. ATR phosphorylates ChK1 at site S317 and S345 to start a slow IR-induced S-phase checkpoint response. ChK1 phosphorylates Cdc25A for degradation ( Shiloh, 2001 ).
Chk2 kinase is responsible for Ser20 phosphorylation which is a key regulator of G2/M checkpoints.
Initiation of DSBs after Chk2 activation is depended on phosphorylation of Thr68 which is ATM mediated.
Fig 3 : Control of cell cycle checkpoints BY ATM and ATR occurred by different types of DNA damages or stalled replication forks.
( a ) : In the G1/S phase p53 becomes activated and phosphorylated because of a series of post-translational modifications mediated by ATM and ATR either directly or indirectly. ATM and ATR directly phosphorylate Ser15 and p53 and Chk2 which is activated by ATM, phosphorylates Ser20. Other pathways also activated by ATM which cause additional modification of p53 and the principle mediator is phosphorylated which is a main regulator of p53 degradation, Mdm2. By this control and stabilization of p53 is achieved strongly.
( b ) : In the G2/M pathway Chk1 and Chk2 phosphorylate by activated ATM and ATR. Generally inactivation of Cdc25C mediates arrest of G2. Phosphate activity of the Cdc25C usually activates the cycline-dependent kinase Cdc2 and cause entry to mitosis.
Two checkpoints are handled by ATM through phosphorylation of Chk2. These two pathways are inter-connected at p53 junction. Different products of p53 activated genes have effects on development of G2 phase. Thus ATM can hold each of the two checkpoints either through p53 or by Chk2 ( Shiloha ). Some evidences shows that Ser15 phosphorylation is not essential for the stabilization of ATM-dependent p53 which is induced by DNA damage, whereas previously it was demonstrated that phosphorylated Ser15 and p53 are associated after DNA damage. This evidence suggests that some other modified ATM-mediated protein is more important for this process. This protein might be MDM2 which is a known oncoprotein. MDM2 acts as a negative regulator for p53; it binds with the amino terminus of p53 and represses its transactivation activity. ( ali ) Apart from phosphorylation of Ser20, Ser376 gets dephosphorylated, which is ATM mediated modification of p53.
As per view, ATM and ATR function in a similar way as both kinases could phosphorylate Ser15 of p53 in vitro. Commonly ATM- dependent phosphorylation is occurred by IR while UV radiations cause ATR- dependent phosphorylation; however same type of damage can be detected by both of them. ATM first activated in response to DSBs and then ATR gives support to phosphorylation of targetd substrates of ATM. Phosphorylation of Ser345 of the Chk1 kinase responds to DNA damage or replication, by which ATR is activated. Thus conclusion can be withdrawn that G2/M checkpoint is activated against any damage by joint and balancing mechanism of ATM-ATR ( Refer Fig. 2 ). p53 is phosphorylated on Ser15 and Ser37 by DNA-Pk in-vitro, but it is not necessary to get phosphorylated for p53 which is activated and accumulated by damage. ATM, ATR and DNA-Pk fall into protein family. Cell cycle progressions, response to DNA damage and genome stability are controlled by members of this family. Severe combined immunodeficiency (SCID) is seen in mice because of DNA-Pk deficiency which has similar characters with A-T.
Another PI3K related protein is DNA-Pk, which can also phosphorylate many proteins including p53 and Chk2. DNA repair has been done by DNA-Pk via nonhomologous end joining (huijun zhu). IR or UV induced DNA damage activates DNA-PK. DNA-PK is activated at primary DNA damage as a sensor rather than downstream effecter of DNA damage signalling. DNA-PK is present in the nucleus at high level and maintains its activity throughout the cell cycle. DNA-PK may be activated by the interaction with DNA and other proteins. In a similar way DNA-PK is also activated by protein-protein interaction. Ku(protein that binds to DSBs and required for non homologous end joining) presents the DNA-PKcs to DNA and modulates the DNA-PKcs-DNA interaction. DNA-PKcs gives its catalytic charge to DNA-PK complex. It leads to that DNA-PK can be activated in the absence of Ku also ( Yang, et al. 2003 ).
2.5 DNA-PK and p53 - formation of close contact
DNA damage is also indicated by DNA-PK with the help of p53 and thus form a protein complex. This protein complex act as a sensor complex which binds to abnormal DNA structures, thus detects the breakage in DNA replication. Also in the DNA-PK mediated pathway, p53 act as effecter ( Yang, et al. 2003 ).
3.0 Aim of the research study
To utilise dot blot and western blot techniques to seek evidence for DNA damage in cryopreservation of lymphocytes by using different concentrations of glycerol.
Hypothesis : If DNA damage is occurred by cryoprotective agent then higher concentration of cryoprotective agent cause more DNA damage. this DNA damage will be analysed by two protein detecting techniques, dot and western blot as DNA damage checkpoints come under protein kinase family.
4.1 Isolation of lymphocytes
Upon receipt of SIGMA- ALDRICH, lymphocytes were isolated from donated human blood using histopaque ( Sigma- Aldrich Histopaque- 1077 ). Boyum in 1968 had described procedure for isolation of mononuclear cells from circulating blood and bone marrow. Histopaque is a mixture of polysucrose and sodium ditriose which has a 1.077 + 0.001gm/ml density. Mononuclear cells are easily isolated from small volume of red blood cells in this medium.
3ml histopaque was taken in 15ml conical centrifuge tube and brought to room temperature. Add 3.0ml of whole blood onto the Histopaque from side wall of the centrifuge tube. Radius of the centrifuge spindle is measured from the centre to the end of the test tube carrier to fix the rpm speed of the centrifuge. Total length of centrifuge spindle was 15cm. According 400 X g, by using nomogram the speed was set on 1500 rpm for 30 minutes at room temperature. During centrifugation erythrocytes and granulocytes are aggregated into the polysaccharide and rapidly settle down the bottom. Lymphocytes and other mononuclear cells remain in the histopaque. Contamination of the erythrocytes is very less. Platelets are aggregated in the upper layer (plasma). After centrifugation upper layer was aspirated carefully by Pasteur pipette. The opaque interface between plasma and histopaque which contain lymphocytes was transferred into other clean tube. 10ml of isotonic Phosphate Buffered Saline (PBS) solution was added into this tube and mixed by gentle aspiration. According to 250 X g the solution was centrifuged at 1200 rpm for 10 minutes. By repeating the steps, aspirate the supernatant and discard. Cells were resuspended again with 5.0ml PBS solution and mixed by gentle aspiration. Cycle was repeated again to 250 X g with 2ml PBS. Before centrifugation solution was split into 4 tubes for 4 different concentration of glycerol. Lastly cell pellets were resuspended in 0.5ml PBS in each tube.
4.2 Treating with cryoprotective agents
4 tubes were labelled as control, 10%, 15%, and 20%. Add 1ml cryoprotective agent according to concentration in each tube except control tube and incubate them at room temperature for 30 minutes. Centrifuge them at 250 X g for 10 minutes. After removing cryoprotective agent add 1ml PBS solution and incubate for 10 minutes at room temperature. By repeating the steps again, centrifuge the tubes at 250 X g for 10 minutes. Repeat the whole cycle once.
Cells were lysed in buffer containing 50mM Tris-HCL (pH - 8.0), 150mM NaCl, 4mM EDTA, 1% Triton X-100, 0.1% sodium dodecyl sulphate (SDS), 1mM dithiothreitol and 1mM phenylmethylsulfonyl fluoride. After adding 1ml lysis buffer samples were frozen.
4.3 Protein Quantification
Protein quantification is often required before proceeding with protein samples for isolation chromatographic or electrophoretic analysis or immunohistochemical methods.
Protein quantification was done by Bio-Rad protein assay. 96 well 300Âµl plate was used. This application is based on dye binding assay. Brilliant blue G-250 dye binds to basic and aromatic amino acid residues, especially to arginin. Ranges of standard solution falls between 0-2 mg/ml prepared with BSA. In each well 5Âµl of either standard or sample was added with 200Âµl of Bio-Rad Assay sample B and 25Âµl of Bio-Rad sample A. The whole preparation was incubated for 15 minutes at room temperature. Blue colour was developed. Density of the blue colour was read by microplate reader. Maximum of dyes absorb at 740nm. A typical standard curve was plotted to calculate protein concentration. According to that concentration samples were taken to perform protein precipitation by methanol- chloroform technique. For this technique total protein sample was dispensed into an Eppendorf tube and the final volume made up to 400Âµl by adding distilled water. In this preparation mixture of 400Âµl methanol plus 100Âµl of chloroform was added. Reagents were mixed and centrifuged for 2 minutes at 10,000 rpm. Protein layer was separated between the chloroform and methanol layer. After removing the upper methanol further 400Âµl of methanol was added and centrifugated at 13,000 rpm for 2 minutes. Pellets of protein were obtained and Eppendorf tubes were air dried. Approximate after 10 minutes pellets were resuspended in 4Âµl of lysis buffer.
4.4 Dot Blot Technique
Dot blot technique was performed with Amersham Hybond-P PVDF membrane. After cutting the membrane to appropriate size, marking on the membrane was done by pencil for different concentration of sample application. Minimum distance 1cm was taken between 2 dots. After that membrane was wet in methanol. 1x5 minutes wash with Millipore water had been done followed by TBS. Then after membrane was placed on a Hybond blotting paper which was soaked into TBS. Care was taken in order to control drying of membrane. Following to this procedure, 4Âµl of each sample ( precipitated protein samples ) was applied on to the membrane and left to air dry.
After air dry, membrane was blocked with the blocking buffer contain 5% milk TBST for 2 hours and then treated with two primary antibodies ATM ( Santa Cruz Biotechnology - 1B10 ) and P-ATM ( Santa Cruz Biotechnology - 10H11. E12 ) separately with 2.5% milk TBST for overnight. Three wash with TBST ( each for 10 minutes ) was done on the next day to remove primary antibody. After completing the wash membranes were treated with secondary antibody - peroxidise labelled anti - mouse antibody ( Vector Laboratories Inc. - CA 94010 ) in 2.5% milk TBST. Incubation of the membrane was done with secondary antibody for 2 hours. Again membranes were washed with TBST for three times. Chemiluminescent technique was used for developing the membrane and they were scanned with help of Typhoon scanner.
4.5 Western Blot technique
In Western Blot technique sample proteins are separated by using SDS polyacrylamide gel electrophoresis (SDS-PAGE) before they are immobilized on the PVDF or nitrocellulose membrane. Separation on nitrocellulose membrane provides information about molecular weight of sample proteins.
4.5 (a) Electrophoretic Separation
For western blot 10% resolving gel and 4% stacking gel were prepared for separation of proteins. After the gel was set, it was placed into the gel holder and then put into the tank. Tank was filled with 1 X SDS-PAGE Buffer ( Electrode Running buffer) (diluted from a 10X stock prepared with containing 30.3 g tris base, 144.0 g Glycine and 10.0 g SDS dissolved in 1liter water. After that protein samples were loaded into the well. With a 10 well mini gel, 30Âµl of protein sample was added in each well. For preparing 30Âµl of sample protein, 20Âµl of protein and 10Âµl of sample buffer containing Î² mercaptoethanol ( 5% of total volume ) were mixed. Lid was placed on the tank and the whole apparatus was plugged with power supply. Apparatus was run at 45V until the samples were passed the stacking gel ( around 1 hour ). At this voltage samples were separated and molecular marker was used to measure the endpoint of electrophoresis.
4.5 (b) Electrophoretic Transfer to PVDF Membranes
After cutting the eight filter papers to appropriate size, PVDF membrane was cut. PVDF membrane was pre wetted in methanol for 10 seconds. Filter pads were soaked in PVDF Transfer Buffer containing 1.450 g glycine ( 39 mM ), 2.900 g tris base ( 48 mM ), 0.185 g SDS ( 0.037% ), and 100.00 ml methanol ( 20% ). Membranes were assembled to prepare Sandwich. First four blotting papers were arranged one on other. One care was taken in order of removing bubbles. Then membrane was placed on those blotting papers. Gel was put on the membrane and again remaining four blotting papers were positioned on the gel to complete the sandwich preparation. The whole sandwich was put into transfer assembly and power was supplied for 45 minutes.
After completing the SDS-PAGE and electron transfer membrane was blocked with 5% blocking buffer containing milk in TBST and rotated for 2 hours. After blocking, membrane was incubated with two primary antibodies for overnight at 40C in 2.5% milk in TBST. Next day primary antibodies were removed and given three room temperature wash with TBST. Again membrane was reblocked with secondary antibody in 2.5% blocking buffer. The whole preparation was incubated for two hours. Again antibody was removed and followed by three TBST washes at room temperature. After then membrane was treated with chemiluminescent reagents to determine the final results and scanned in Typhoon scanner.
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Fig 4: separation of lymphocytes.
Lymphocytes were separated from whole blood cells using density gradient medium technique. In density gradient technique different blood cells are arranged according to their density. In this medium each component has a different density and with that particular density, it acts as a selective barrier for every type of cells. If the density of cells is higher than the medium it will penetrate through the medium during centrifugation. However if the density is lower than the medium it will form an upper layer on the medium. Density of HISTOPAQUE-1077 is 1.077 g/ml while density of lymphocytes generally falls between 1.065-1.075. Therefore lymphocytes make an upper layer on histopaque. (Fig. 4)
Fig. 5 : BIO-RAD assay
Fig 6 : Protein quantification Graph
Table 1 : Values from microplate reade for protein Quantification
This table shows the values for standard samples and the protein samples of lymphocytes. As per seen in the table protein concentration is decreased as increasing the concentration of cryoprotective agent. This indicates that DNA damage is increased with increasing the concentration of cryoprotective agent. As DNA damage is more, ratio of cell death is more which is resulted in decreasing the protein concentration.
We can determine concentration of solubilised protein by using simple and accurate BIO-RAD protein assay which is based on the method of Bradford. Bio-Rad Protein Assay is also known as a dye-binding assay in which dye changes its colour according to different concentration of protein in sample. According to Beers' law, accurate protein quantification has been done by selecting an appropriate ratio between dye volume to sample concentration. Main principle of this assay is based on colour change of Coomassie dye according to different protein concentration. Mainly basic and aromatic amino acid with polypeptide chain binds with this dye. This assay provides ready-to-use handiness when dye was taken at 1X concentration and 6 protein samples were taken into 0-2 milligram concentration. Three known forms of dye are available: (a) cationic dye gives red colour. (b) anionic dye gives blue colour and (c) neutral dye gives green colour. When protein sample was bounded with the dye, the dye was converted into stable unprotonated blue form. This blue protein-dye complex was detected at 740nm by using micro plate reader.
How to select a protein standard? To use a purified preparation of protein is the perfect idea for standard protein samples. The protein which gives similar colour to the sample being assayed is the best relative standard. Bovine Serum Albumin (BSA) and gamma-globulin are most commonly used as standard protein. BSA was used as a standard protein as colour development by dye is significantly greater with BSA than other proteins.
As per Fig.5 standard curve was drawn by plotting the absorbents value for 740nm on Y- axis and their standard concentration in Âµg/ml on X- axis. According to those values concentration of samples were calculated and further used for protein precipitation by methanol- chloroform method.
Fig. 7 - Dot Blot strip showing result with P-ATM antibody with 20% concentration of glycerol.
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Fig. 8 - Dot Blot strip showing result with ATM antibody.
After getting triplet readings with sufficient samples, statistical analysis can be achived. ANOVA or student T-test can be used for statistical calculations. GraphPad Prism software can be more useful with more dataset and P-values can be calculated.
Various cellular responses can be measured by cryopreserved lymphocytes. Moreover lymphocytes are good source of DNA (Victoria, 2003). When a solution is cryopreserved, intercellular or extracellular ice is formed and the fluid becomes more hypertonic. In case of blood, when it freezes, the extracellular fluid becomes hypertonic and therefore water flows out of the cells. On account of this cell volume reduces. It is believed that due to this, damage occurs either by intracellular dehydration or by stress on cell membrane. Glycerol is thought to gain its effect as a cryoprotective agent by increasing the unfrozen protein at lower temperature and in that way ionic composition is reduced (Morris, 2001).
Some research studies indicate that cryopreservation has some disadvantages in that it causes cytoplasmic damage which may limit its uses. It is important to understand the cellular response during DNA damage. DNA fragmentation occurs because of process of cryopreservation (Thomson, 2005). The current study was intended to investigate this possibility further by focusing on DNA damage checkpoints which were function as sensors for DNA damage. ATM and ATR protein kinases are the key regulators of DNA damage responses (R & D). Cryoprotective agents mostly affect on DNA strands and cause strand break ( Kopeika, 2003 ). DNA double strand breaks primarily moderated by ATM (Michiyoshi, 1999). Therefore in this research project, the main focus was to develop a methodology to monitor activation of ATM and so to indirectly identify DSBs in DNA of human peripheral lymphocytes.
ATM is present as a dimer or oligomer in undamaged cells. ATM is known to be silent in undamaged cells because of its association with FAT region or another ATM monomer. ATM autophosphorylates or transphosphorylates on residue Ser1981 after formation of DSB and gets converted into active ATM monomer from inactive ATM dimers. After activation of phosphorylated ATM molecule, ATM interacts with and phosphorylates other downstream proteins. In turn these have an effect on more than one cell cycle checkpoint (Abchem). Phosphorylation of ATM suggest that DNA damage occur during the earlier stage before genomic instability takes place and wild type p53 loses its activity (Pusapati et al, 2006). As seen before, ATM is phosphorylated on Ser1981, by using an antibody which is reactive with the phosphorylated Ser-1981 ATM epitope, phosphorylated ATM can be detected. According result (fig 6), it is concluded that the antibody which I had used as a primary antibody P-ATM was certainly specific towards phosphorylated Ser-1981 epitope to ATM. My result thus supports the study of Akira et al. (2005).
The main aim of my research work was to monitor DNA damage in cryopreservation of lymphocytes by using different concentration of glycerol as a cryoprotective agent. I had used three different concentrations which were 10%, 15% and 20% of glycerol. According to figure 6, result was obtained for 20% glycerol. It indicates that DNA damage was occurred at 20% concentration of glycerol and phosphorylated ATM was released. It means that DNA damage was not occurred in lower concentration than 20%. This result is supported by Rapataz et al., who also treated human blood with 10% glycerol to preserve erythrocytes in 2006. They showed that injury was the minimum with 10% glycerol. In one another research work, DNA damage was obtained at 10% DMSO ( Williams, personal communication). This result supports the statement of Meryman ( 1971 ) that glycerol has less toxic effect than DMSO.
The western blot technique needs more optimization as appropriate results were not obtained for that. There are many possibilities for not obtaining results. The one most significant possibility is insufficient transfer time. Transfer of protein from gel to membrane is one of the important steps in western blot technique. Large molecular weight proteins require a longer transfer time. ATM is a large protein containing 350 KDa weight. In this case, care should be taken by increasing the electrical current or increase the time of transfer. Moreover care should also be taken in order to avoid formation of bubbles between the membrane and the gel.
One other possibility is might be buffer components were not proper or might be methanol content was very high than required. According to standard protocol, 25ml methanol is required in order to prepare 1 litre transfer buffer. In addition to that, there is one another possibility related with PVDF membrane. It was possible that PVDF membrane became dry in order to transfer protein properly.
Moreover there are some other troubleshooting possibilities linked with protein samples also. Amount of loading protein sample is also an important prospect to get results. 30Âµg of protein samples were loaded in each well according to standard requirement but might be there was inadequate protein concentration in the sample.
There are some other risks related with antibodies. One possibility was that concentration of primary or secondary antibody might be high than required. Moreover primary and secondary antibody should be well suited with each other. In this case, secondary antibody might not compatible with the primary antibody. In addition primary antibody might not be recognised by sample protein. By crosschecking this possibility with dot blot result, conclusion is drawn out that ATM antibody was not appropriate for the sample protein (Fig. 7). Cross reaction might be occurred between blocking agent and primary or secondary antibody.
Additionally protein samples give chemiluminescent signals for very short time and scanner was taking more time for scanning the gel. Therefore signals might not be detected by the scanner. This can also be considered as one strong possibility. This condition can be defeated by using an X - ray film because it is not concern with the enzyme substrates but just dark regions are developed which match with the protein bands of interest.
These results indicate that after doing crypreservation of lymphocytes with glycerol, it is concluded that DNA damage has occurs with 20% glycerol ( Fig.6). Lymphocytes can be successfully cryopreserved at lower than 20% glycerol like 10% or 15%. With 20% glycerol DNA stands break at endogenous level ( duthie) and released phosphorylated ATM and detected by P-ATM antibody which was very sensitive towards phosphorylated ATM.
As seen from the discussion Western Blot technique needs more optimization. In future Western Blot can be more optimized by using other antibodies or by taking more amounts of samples. One other option is different alternative techniques can be used for Western Blot. One technique can used as alternate for Western Blot is RNA aptamer - functionalized quantum dots. This technique has application in protein quantification. The other alternative method is six - histidine ( 6 - his ) tag technology in which according to the code of protein 6 histidine are added. By using either nickel or cobalt column resin proteins can be easily detected by this technology. With this technology efficiency has greatly increased for the targetd protein isolation. Some other techniques can also be used as a alternative for Western Blot are ELISA and Multi - Array platform.