- Material and Methods
Histone Proteins are favourably the alkaline proteins which are present in the nucleus of the eukaryotic cell. They are the basic parts responsible for wrapping and organizing DNA into chromosomes in the nucleus. They are the principal protein elements of the chromatin and acts as a spool around which the DNA winds. In the nucleus of the cell the DNA is arranged in a very dense and super coiled style to form chromosomes. This is referred to as DNA packaging. (Isenberg, 1979) The unwound DNA in the chromosomes is very long and cannot be fit in the nucleus of the cell, hence these histone proteins brings about the DNA wrapping thereby reducing the dimensions of DNA in the cell. (Alberts B et al., 2001)
Histones are primarily categorized into five major types. They are H1/H5, H2A, H2B, H3 and H4. They are categorized into two classes. They include core histones with H2A, H2B and H3 and linker histones with H1 and H5. Two of each of core histones unites to form one octameric nucleosome which is the fundamental sub unit of chromatin. (Bartova et al. 2008; Bonisch et al. 2008). Hence histones proteins form the nucleosomes directly. After extraction of chromatin from the cells it is like beads on string. The string is the DNA and the beads are the nucleosomes which are roughly disc shaped. The linker histone H1 separates the nucleosome with the other nucleosomes and also helps in bringing the adjoining nucleosomes for further super coiling. The octameric nucleosome is formed by two H2A, H2B dimers and a tetramer of H3, H4 histones. These core histones are comparatively analogous in structure. However their 3D structures are relatively different.
The core histones have different protein sequence. The sequence for H2A Histone is SGRGK QGGKA RAKAK SRSSR AGLQF PVGRV HRLLR KGNYS ERVGA GAPVY LAAVL EYLTA EILEL AGNAA RDNKK TRIIP RHLQL AIRND EELNK LLGRV TIAQG GVLPN IQAVL LPKKT ESHHK AKGK. (Andreas and David, 1998)
The sequence for H2B Histone is PEPAK SAPAP KKGSK KAVTK AQKKD GKKRK RSRKE SYSIY VYKVL KQVHP DTGIS SKAMG IMNSF VNDIF ERIAG EASRL AHYNK RSTIT SREIQ TAVRL LLPGE LAKHA VSEGT KAVTK YTSSK. (Andreas and David, 1998)
The sequence for H3 Histone is ARTKQ TARKS TGGKA PRKQL ATKAA RKSAP ATGGV KKPHR YRPGT VALRE IRRYQ KSTEL LIRKL PFQRL VREIA QDFKT DLRFQ SSAVM ALQEA CEAYL VGLFE DTNLC AIHAK RVTIM PKDIQ LARRI RGERA. (Andreas and David, 1998)
The sequence for H4 Histone is SGRGK GGKGL GKGGA KRHRK VLRDN IQGIT KPAIR RLARR GGVKR ISGLI YEETR GVLKV FLENV IRDAV TYTEH AKRKT VTAMD VVYAL KRQGR TLYGF GG. (Andreas and David, 1998)
The molecular weight of the core histone proteins are measured in the Daltons. The theoretical molecular weight for H2A histone protein is 13,990.28 Daltons, for H2B histone protein is 13,788.97 Daltons, for H3 histone protein is 15,272.89 Daltons and for H4 histone protein is 11,236.15 Daltons. (Kornberg, 1977;McGhee and Felsenfeld, 1980)
The histone proteins play a significant role in chromosome stabilisation, gene regulation and expression. The main functions of histone proteins are condensing the DNA strands and chromatin regulation. These histone proteins form the nucleosome and they impart a structure to which DNA is twisted and causes to fit the bulky genomes of eukaryotes inside the nucleus of the cell. (Bartova et al. 2008; Bonisch et al. 2008). The histones also undergo post translational modifications which alter their interaction with DNA and nuclear proteins. The H3 and H4 histones have very long tails projecting from the nucleosome which can be covalently altered at numerous places and the alteration includes the acetylation, ubiquitination, methylation, phosphorylation, ADP-ribosylation and citrullination.(Bartova et al. 2008; Bonisch et al. 2008). The modification of histone includes the gene regulation, DNA repair and chromosome condensation. In this manner, histones can control gene expression, cell growth and proliferation. (Jenuwein and Allis 2001).
The separation of the histone proteins is carried out by using the 1D SDS PAGE (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis) method.
In the gel electrophoresis method the charged molecules are segregated based on their physical properties like charge, mass, etc. by allowing them to pass forcedly through a gel matrix by an electric current. (Coligan et al., 2002).The proteins from the various complex protein mixtures are normally separated by this method by using the polyacrylamide gel. It is known as Polyacrylamide Gel Electrophoresis (PAGE method). In this method the main constituent is the acrylamide which is used in the preparation of the electrophoresis gels for the isolation of proteins. In order to form the cross linked polymer network, the acrylamide is combined with bisacrylamide with the polymerising agent like Ammonium Per Sulphate (APS). The polymerisation reaction is catalysed by TEMED (N,N,N,N’-tetramethylenediamine) by the producing the free radicals by APS. The size of the pores and firmness of the gel matrix mainly depends on the ratio of bisacrylamide and acrylamide used and their total concentration. Sequentially these in turn depends up on the range of molecular weight of the proteins which has to be determined. The pore size of the gel matrix is contrary to the quantity of acrylamide used. Generally 12% of polyacrylamide gel has lesser pore size than 7% of polyacrylamide gel. Larger proteins are resolved with the gels with less amount of acrylamide and smaller proteins with more amount of acrylamide. And for the broad range of protein sizes, the special gels like Gradient gels are prepared by having less percent of acrylamide at the top/start and more percent of acrylamide at the bottom/end. The electrophoresis gels are mixed with the buffers which impart the conduction of electric current though the matrix. The solution is transferred to the gel cassette which is a thin space formed by placing two glass or plastic plates facing each other. After the gel is polymerised, the cassette is placed vertically into the electrophoresis tank containing the electrodes. The proteins are added in the wells from the top and electrophoresis is carried out during which the proteins are separated by the gel due to its sieving properties. In order to obtain the best possible resolution of the proteins a gradient gels are used. (Hames et al., 1990)
There are different forms of PAGE available which are used for separation of different proteins based on different principles. They commonly include the native PAGE, SDS PAGE, 2D PAGE, etc. (Hames et al., 1990; Coligan et al., 2002).
In case of the 2D PAGE (Two Dimensional Polyacrylamide Gel Electrophoresis) the proteins are isolated by isoelectric point in the first dimension and later by mass in the second dimension. This method offers the maximum resolving power for the protein analysis and it is a very significant method in proteomic research. (Hames et al., 1990)
In the native PAGE, the proteins are isolated based on their size, net charge and shape. The electrophoretic migration is due to the fact that the most of the proteins possess a net negative charge in alkaline running buffers. As the negative charge density increases, the migration speeds of the protein increase. Simultaneously the frictional force of the gel matrix produces a sieving effect thereby reducing the protein movement based on their size and shape. Hence the smaller proteins face less frictional force and larger proteins face more frictional force. In this manner the various proteins are separated from a mixture. (Hames et al., 1990)
In case of the SDS PAGE the gel is mixed with the buffer having the SDS (Sodium Dodecyl Sulphate). The SDS is heated with the proteins samples before electrophoresis so that the charge density of all proteins are almost equal. The SDS is an anionic detergent which denatures the proteins present in the sample and attaches strongly to the uncoiled molecule. In order to make sure that no quaternary or tertiary proteins structure remains generally a reducing agent like dithiothreitol (DTT) is also added as it ruptures the protein disulphide bonds. Hence when these samples are used in the electrophoresis, the proteins get separated based on their mass alone. In this method a set of proteins of known molecular weight is run aside the sample in the same gel cassette. They act as a reference from which the mass of the sample proteins is determined. They are called as Molecular Weight Markers. The electrophoresis is carried out by using the two gels for favourable results. They are the stacking gel and the resolving gel. The stacking gel is added over the top of the resolving gel. Apart from the low concentration of acrylamide in stacking gel, it has low pH and different ionic content than the resolving gel. This causes the proteins to get concentrated into a tight band during the early electrophoresis period before entering into the resolving gel. (Hames et al., 1990)
Breast cancer refers to the hysterical growth of breast cells. The breast carcinoma is a malignant tumour. It occurs due to any abnormal genetic changes in the breast cell. Presently there is an increasing alarm regarding the high risk posed by various compounds with oestrogen like activity present in the environment. These various compounds includes the Phytoestrogens (e.g. genistein), food products like legumes, lentils, chickpeas, soybean, cereals, fruits, and vegetables, industrial contaminants, (e.g. bisphenol A) and polychlorinated biphenyls, organochlorine pesticides (e.g. endosulfan), etc. These substances have the ability to produce cancer mediated through estrogenic receptors. Some of these substances when treated with the MCF-7 cell line, MCF-10F (ER??/ER?+), MDA-MB-231 (ER??/ER?+) or MCF-10A (ER??/ER??) cells, increased the growth rate of these cell lines. This 3-4 folds proliferation was due to the up regulation of all the core histone proteins which represents the increase in the chromatin content of the cells. The degree of proliferation of the cells indicates the level of the core histone proteins and was concentration dependent. Hence this indicates that histone proteins are used as indirect markers of breast cell proliferation. Therefore in the human breast cancer the histones are up regulated and cause cell proliferation mediated by the oestrogen receptors agents. Based on this it is possible to state that the core histone proteins serve as bio markers of (ER+) human breast cancer.(Zhu et al., 2009)
The main aim of this experiment is to resolve the given four human recombinant protein samples (H2A, H2B, H3 and H4) by using the one dimensional SDS Poly Acrylamide Gel Electrophoresis (1D SDS – PAGE).
Material and Methods
The experiment uses the Bio Rad Mini PROTEAN 3 Cell gel electrophoresis unit. It consists of the electrophoresis tank, electrode chamber with a cathode and anode, two glass plates, gel cassette assembly, comb and the connecting cables. Power source, heater, micro centrifuge, pH meter, and the other general equipments like test tubes, conical flasks, etc were used.
The various reagents used in this experiment includes the Acrylamide/bisacrylamide (30%w/v/0.8%w/v), 3.0M Tris/HCl (pH – 8.8 for resolving gel stock), 0.5M Tris/HCl (pH – 6.8 for stacking gel stock), TEMED, SDS (10%w/v), Ammonium Per Sulphate (APS – 25%w/v). The running buffer is also used as 0.025M Tris/0.192M glycine/0.1% (w/v) SDS, pH 8.3. The other reagents used are sample buffer, Pre stained molecular markers, colloidal Coomassie Blue Stain Solution, Bromophenol Blue (0.5%w/v) and the given four human recombinant proteins (1µg/µl) [H2A, H2B, H3.3 and H4].
The SDS PAGE method is commonly used for the examination of the proteins because of its simplicity, speed and resolving capacity. The SDS PAGE is the Sodium Dodecyl Sulphate Poly Acrylamide Gel Electrophoresis method. The various steps performed are as follows:
Preparationof Gel Cassette Sandwich
Firstly select a clean Spacer Plate and Short Plate and place them over one another and set them in the casting frame and lock the pressure cams from both the sides. It should be kept in such a manner that the short plate should face the front of the frame. After locking the gel cassette assembly check that both the plates are flush at the bottom. Then place the rubber gasket at the bottom of the casting stand and place the gel cassette sandwich assembly in the casting stand in such a manner that it is perpendicular to the level surface. After complete formation of the gel cassette assembly, add water into it to make sure that no leaking takes place. Then remove the water and wipe it with the filter paper. Set the comb into the assembled gel cassette and mark the level at 1 cm below the comb teeth and remove it, the resolving gel is poured up to this level. After preparation of the resolving gel pour it up to the mark into the assembled gel cassette in a very smooth way to prevent any air bubble formation. Allow the gel to set for 45 minutes. After the gel has set wash its surface with distilled water and wipe with the filer paper and allow drying the area in between the glass plates for a minute. Then after this prepare the stacking gel solution and pour it above the resolving gel till the top of the plates. Then immediately place the comb in the stacking gel in gel cassette to form the wells. Allow the stacking gel to set for overnight.
Preparation of Gels
The gels used in SDS PAGE method are the resolving gel and the stacking gel. Both the gels consists of the acrylamide/bisacrylamide, distilled water, 10% SDS, TEMED and ammonium per sulphate (APS). However the resolving gel uses 3.0M Tris/HCl (pH 8.8) and the stacking gel uses the 0.5M Tris/HCl (pH 6.8). These gels are prepared by directly mixing all the reagents. However the TEMED and APS are added only when the gel is ready to be polymerised. In this manner the gels are prepared.
Electrophoresis Module Assembly
After the stacking gel has set, smoothly remove the comb. Then open the cams and remove the sandwich gel cassette and place it in the electrode assembly in such a manner that the short plate faces inward of the electrode assembly. Secure the electrode assembly by closing the two levers of the frame to form the inner chamber. Set this secured inner chamber into the Mini Electrophoresis Tank. Fill the inner chamber with the running buffer, however it is not overfilled. Then add around 200 ml of the running buffer is added into the Mini Tank.
Sample Preparation and Loading
Four recombinant histone protein samples are used in this experiment. They are H2A, H2B, H3.3 and H4. 1µl of each of this protein samples (1µg/µl) is added to 20µl of the Sample Buffer in the Eppendorf tubes and labelled properly. They are heated in a heater set at 100? C for two minutes and then allowed to cool to room temperature. After the sample preparation, 20µl of each of the histone protein is loaded into the wells carefully by using the gel loading tips. The sample loading should be very smooth and the tip should not puncture the well. Load the 2µl of the pre stained molecular marker (Page Ruler) into the other well simultaneously to ensure the movement of proteins.
After loading the histone proteins, mini tank is covered with the lid and the electric supply is given by the cable wires. Turn on the power supply and the start the gel electrophoresis at 200 Volts for about 35 minutes until the bromophenol blue dye has drifted to 1 cm from the bottom of the gel. During this time each histone protein based on their molecular weight is moved to their ends.
Staining the Gels
Once the electrophoresis is completed, switch off the power supply and take out the lid. Cautiously lift the inner chamber assembly and dispense the running buffer to avoid the falling of buffer before opening the cams. Then open the cams and take out the glass cassette sandwich. Open the sandwich with the help of gel releaser and take out the gel slowly. Immerse the gel in 30 ml of Colloidal Coomassie blue stain solution for half an hour with moderate shaking. During this time the staining occurs and once it is completed the gel is de stained with water and then incubated with water at room temperature for overnight for complete de staining. After this the gel is rinsed with water for the proteins bands to be seen clearly.
Figure 1: SDS Poly Acrylamide Gel Electrophoresis of Histone Proteins. Bands show the migration of individual histone proteins. A pre stained molecular marker is included at the side for comparison.
In the present experiment four human recombinant histone proteins were given for isolation and determination. From the standard pre stained molecular marker, each of the histone protein has to be determined by correlating with its band. The Figure 1 illustrates the SDS Poly Acrylamide Gel Electrophoresis of given four recombinant histone proteins (H2A, H2B, H3.3 and H4). In the figure the last column of bands refers to the standard pre stained molecular marker (Page Ruler) and the other four bands represents the respective histone proteins. The values beside the molecular marker indicate the molecular weight in kDa. Hence with reference to this standard values, the four given histone protein samples has to be determined. In case of the band of H2A histone protein, it is nearly 13,990 Da as it is below 15 kDa. Similarly in case of the band of H2B histone protein, it is nearly 13,789 Da. And in case of H3.3 histone protein, the band is clearly at the top of all the bands indicating it has the highest value and it is slightly above 15, so it may be around 15,273 Da. In the fourth histone protein H4, the band is at the far end when compared to the other three bands. However it is slightly above 10. Hence it may be around 11,236 Da. Therefore the molecular weights of the histone proteins were identified. However the standard molecular weight for the H2A is 13,990.28 Da, for H2B is 13,788.97 Da, for H3.3 is 15,272.89 Da and for H4 is 11,236.15 Da. (Kornberg, 1977;McGhee and Felsenfeld, 1980)
The separation of proteins is mainly performed by the gel electrophoresis method. There are different types of gel electrophoresis methods used to separate different types of proteins. However in the present experiment the isolation of histone proteins is mainly carried out by 1D SDS PAGE method. This method is a one dimensional method and it separates the proteins principally by their molecular weight. It makes use of the ionic detergent Sodium Dodecyl Sulphate (SDS). The poly acrylamide gel is mixed with SDS because it denatures the proteins and binds to them to make them approximately evenly negatively charged. This is done with the aid of heating and generally a reducing agent like dithiotheritol (DTT) in order to break the protein disulphide bonds. This removes the risks of presence of any tertiary or quaternary proteins. As a result when electric current is passed through a power supply, all the SDS bound proteins of the sample will drift in the gel matrix to the anode, thereby separating the proteins based on their molecular weight alone. The proteins with low molecular weight moves fast through the gel compared to the proteins with high molecular weight due to the sieving effect of the gel matrix.
Therefore in this experiment a set of four human recombinant histone proteins (H2A, H2B H3.3 and H4) were given to separate them based on their molecular weight. Hence these protein samples were taken and were subjected to the SDS PAGE method. In this method firstly the gel cassette assembly was formed by using the two glass plates (Spacer Plate and Short Plate) in a proper manner such that the short plate is facing front of the frame. After assembling the gel cassette it was filled by the gels which were prepared just before use. Firstly the resolving gel was poured and after it got set for about 45 minutes, the stacking gel was poured and comb was placed in it and it was allowed to set for overnight. The pouring of gels was carried out very carefully and slowly in order to prevent any air bubble formation. The appropriately set gel with the well defined wells is selected and gel cassette assembly was set in the electrode unit in the Mini Electrophoresis Tank. The running buffer was added properly and the sample histone proteins were loaded very cautiously. Before loading the protein samples, they were heated for about two minutes. Along with the samples, the pre stained molecular marker was also loaded. And the power supply was given and the electrophoresis was carried out for 35 minutes at 200V. During this time the bromophenol blue dye moves to 1cm from the bottom of the gel. After this the power supply was switch off and very carefully the gel was removed from the gel cassette sandwich assembly and was dipped in the staining solution like Colloidal Coomassie blue stain solution for about half an hour with occasional shaking. After staining, the gel is removed and de stained with distilled water by incubating it for overnight. Then it was rinsed properly to visualise the bands for each of the histone proteins.
The pre stained molecular marker of known molecular weight was also run beside the given histone samples in the same gel. It acts as a standard or reference. For all the given four human recombinant histone proteins the bands lie in between the 10 and 15 kDa as per the marker. In situation of H2A histone protein, the band obtained is just below the 15 kDa band in the standard. Hence its molecular weight is around 14 kDa. In case of the H2B histone protein, the band is obtained also below 15 kDa but a little lower than H2A, hence it should be less than 14 and should be around 13.7 kDa. In case of H3.3 histone protein, the band is just above the 15 kDa and it should be around 15.3 kDa and for the H4 histone protein the band is quite low and just above the 10 kDa band; hence it should be around 11.3 kDa. The data obtained in this experiment is fairly correct as compared to the standard theoretical data. The theoretical molecular weight for H2A histone protein is 13,990.28 Daltons, for H2B histone protein is 13,788.97 Daltons, for H3 histone protein is 15,272.89 Daltons and for H4 histone protein is 11,236.15 Daltons. (Kornberg, 1977;McGhee and Felsenfeld, 1980). Hence the results obtained in this experiment are reasonably acceptable and can be used for further studies.
If the mixture of these histone proteins were need to be separated by the same method (SDS PAGE), then the expected result would not be accurate and proper. And the band for the individual histone protein may not be clear. It is because all the given four human recombinant histone proteins have approximately equal and very close molecular weights. And the values for all the four histone proteins lie between 10 to 16 kDa. Hence the bands would be very close to each other. If any flaws or any possible inaccuracy occurs during the experiment, then there is a possibility that the bands may be wrong. Hence the results would be wrong. Therefore there is very less possibility that the results would be accurate and also for the correct identification of the bands. However, if the experiment is carried out perfectly, then in such case the order of bands would be H4, H2B, H2A and H3.3 in the increasing order of their molecular weights. It is resolved with the help of the standard molecular marker which acts as a reference. Therefore, if mixture of these four histone proteins were to be separated by this method very accurately, then the results may be correct. However, for the separation of this mixture of four histone proteins another laboratory method is available. In this method, the groups (H2A, H2B) and (H3, H4) are separated first by using NaCl and hydroxylapatite chromatography. (Richard and Gary, 1979) The H2A and H2B group are the slight lysine rich group and H3 and H4 is the arginine rich group. This method was introduced by Van der Westhuyzen and Von Holt. In this method, firstly the individual groups were separated. However, the given mixture was treated with 0.63 M NaCl and 0.1 M potassium phosphate before the mixture sample is loaded on to the hydroxylapatite. This is to remove any contamination with the other linker histone proteins like H1 and H5. Then it was subjected to chromatography to obtain the core histone groups (H2A, H2B); (H3, H4). After this the samples obtained are subjected to SDS PAGE method for further separation. Finally the core histones were separated. (Richard and Gary, 1979)
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