Biomarker Discovery Estrogen Stimulating Human Breast Cancer Cells Biology Essay

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1975 Southern introduced the method for identification of DNA from gel electrophoresis called as southern blotting method. Parallel to it, there is a method to identify the DNA which was known as northern blotting. In years 1979 this blotting technique was called as western blotting technique. Year 1979-1980 says that, "electrophoresis proteins" can be moved to nitrocellulose can be analysed by nitrocellulose using various probes such as lectin and different antibodies.

Basic criteria present in the gel electrophoresis consist of two different methods

One dimensional method.

Two dimensional methods. (Dunbar, 1996)

Methodologies used in protein separation

Introduction

Mass spectroscopy was mainly used prior to two dimensional electrophoresis

Proteomes is the term used for separation of proteins. It was put forth by "Wasinger et al 1995". Proteomes terms mean entire complement of proteins expression by genome.

Two dimensional and one dimensional method are the two different methods used in the separation of proteins. Amongst these methods, two dimensional methods are sayed to be the standard method. It occurs under the denaturing condition with subsquential mass spectroscopy analysis. (Westermeier and Schickle, 2009)

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Main mechanism behind the electrophoresis.

Isoelectric focusing point.

It is the point where ion are separated according to their ionic charge, which are being applied to them in presence of the electric field .Stable ph gradient is created in the time of isoelectric focussing, at anode and cathode .It is the electric point at which molecule or particle does not carry any charge . It's the particular ph at which this state occurs, such type of points can be said to be ISOELECTRIC POINT or "pI". (Melvin, 1987)

Preparation of poyacrylamide gels

Preparation occurs due to cross-linking acryl amide with N, N' -methylenebisacrylamide. (Melvin, 1987)

Methods

The first dimensional method.

The second dimensional method.

The ONE dimensional method

Originally gel rod where used through which ph gradient is generated. O 'Farrell (1975) was the first person to introduce the technique, which was depending on the immobilized ph gradient known as IPG. In one dimensional method IPG strips are used, which can be easily handle then Gel rods. Result can be obtained faster due to fixed gradient present in the gel matrix.

Mainly the one dimensional method shows different ways of sample loading .it is found to be difficult in Sample loading in the one dimensional than two dimensional method.

Strips should be rehydrated first then they can be used for first dimensional process.

Concentration on each compounds may varies slightly .Two ways of sample loading on the strips .first mixing sample in rehydrates solution swelling occurs into gel. This process is called loading on pre-rehydrated strip at anode or cathode ends of gradients , the cup or paper bridge model which can be seen in figure below.

Fig as shown below

Fig {{30 Wulfkuhle, Julia.D.Liotta, Lance.A.and Petricoin, Emanuel.F. 2003 ;}}

One dimensional method proves to be more critical method.

Showing Disadvantages

1. Proteins are lost in sample loading.

2. Sample loading is critical than two dimensional method.

3. So result is highly influenced.

4. Rehydration causes accumulation of proteins with unequal charges sign.

So that proteins carry very low charge which leads proteins to stay away from IPG strips.

Ph gradient should be only run on the anode side.

Ph gradient is needed to be determined for each solution.

Another Disadvantage of one dimensional method is instrumental assembly, needs about 12 IPG strips with inter cooling. But at commercial level there is no instrument which can have different electric field on each strip along with controlling them. It is because little difference in conductivity on strips may cause the results to vary.

Solution: horizontal strips streaking can be another reason for reduction in ph gradient reductant used is DTT .it's a basic reducatant. Presence of alkaline environment makes DTT negative charged which leads it to move towards anode, this is the main reason for cystein to be unprotected proteins. And cause then to saturate.

It can be avoided by changing the solution with HED hydroxyethylsulfacte at the anode side; cystein remains stable due to oxidisation. Good possible result is obtained. "Olsson et al, 2002"

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Paraffin oil, protects strips from oxygen. Temperature actively running at 20 degrees.

Two dimensional method

It is continuous step to one dimensional method , next steps involves protein separation focusing of proteins to IEF , strips need to be equilibrated for 2 times for 15 min in an buffer solution with 2 % of SDS , urea , 30% glycerol , 1% DTT , 2 -5 % idoactetamide in second step containing SDS - PAGE. Standard procedure includes 1.5mm thick gel slab of glass with homogeneous mixture of gel. They are needed to be polymerized in between two glasses. Some cases need improvising of glycoprotein's size which can be done with the help of porosity gradient gels. After applying gels strips at the end into buffer tank.capicity may varies from 7 to 24 l buffer its tris chloride of ph 8.8. Especially "Laemmli (1970)" described the running buffer composition including tris, chloride, SDS and glycen.

Now a day's two dimensional electrophoresis previously described by "O Farell (1970)", absence of stacking gel, due to protein pre-separation. The spot picking is needed for mass spectroscopy, which requires gels to be covalently fixed to one of glass plates. Gel containing laemmli buffer should not be stored more than 4 weeks, due to polyacrlyamide gel containing buffer. Ph does not have long shelf life due to alkaline hydrolysis. So it is needed to be rebuilt.

There are various solutions to take over.

Film backed gel: varies in size of gel, put forth by "G E healthcare". Ph 6.7 tris acetate.

Vertical gel boxes: invitrogen, ph below 7, i.e. bis tris buffer in gel along with tris MOPS or Trsi MES at cathode buffer.

There is large number of commercial available gels for SDS-PAGE.

Gels placed between glass plates

Composition: Laemmle buffer, ph 8.8, short shell life.

PPA buffer ( piper dine propionamide as a replacement for tris , shelf life is one year due to ph value between 7 . Gels run along with standard laemmli electrophoresis buffer.

Film backing with PPA buffer, used for visible detection technique for example zinc imidazol, coomassie brialliant blue or sliver staining.

Non flour cent film backing for DIGE, semiconductor buffer solution, ph below 7, stability is more.So in order to achieve long shelf life of buffer semidicontinous buffer can be used due to the composition which contains chloride, glycien or tricine ions which are called as leading or trailing ions as compared to Laemmle classical discontinuous buffer. Ions cause stacking movement from tailing and leading ions, which causes ph to adjust below 7.

Instrumenting can be done in two ways

1. Vertical running.

The instrument shows cassettes which are inserted sideways into a common buffer basin where migration is left to right and its divides buffer from top to bottom. But only one buffer can be used. Applicable for multiple phase buffers. System with controlled temperature and low buffer volume.

2. Horizontal set up.

"Gorg et al (1995)" cassettes are not inserted sideways; IPG strips are not present between two glasses. Thinner glass is used in film backed gel. Resolution is more which allows rapid running of gel. Buffer chambers are not present instead of it filter paper wicks are present on both the sides of the gel. Before use they are pre treated by buffer solution.

Detection of proteins.

Visible staining: coomassic brilliant blue, sliver staining, and auto radiography it was previously used, but now post staining technique is used.

Florescent detection: multiple analyses of different sample. But need more equipment like high performance florescent scanners, cameras.

(Westermeier and Schickle, 2009)

Potential approach applied in biomarkers discovery

Following techniques shows potential approach

1.Oncoproteomics and molecular study of tumour cells in cancer.

According to the article, with the introduction of two dimensional method proteomics first came to existence. Widely used mass spectrometry, protein chip, advance bioinformatics, fractional techniques in the identification and treatment of cancer. With the help of oncoproteomics better understanding of cancer pathogens, in development of new tumour biomarkers, early detection and screening of cancer can be carried out. Clinical practice is one of most important factor which can be carried on the bases of molecular cancer study.

Current tumour markers

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Early detection is one of the difficult factors in the detection of the cancer cells, its total due to lack of identification of the symptoms or lack of required symptoms. Markers present in the blood are the blood tumour markers used in breast cancer cells CA cancer antigen 15-3 it is one of the developed markers which is useful in early detection because of its low sensitivity. So measurement of CEA and HER-2 as carcinoembryonic antigen, which do exist in abnormal discharge of nipple excresion, is consider of analysis in several countries.(Cho, 2007)

2. Diagnosis markers

It is the part of oncoproteomics study it involves the diagnosis of malignancies In early detection of cancer, thermos table fraction of serum in breast cancer cell is analysed by two dimensional gel electrophoresis combines with "matrix assistance laser desorption/ionization/time of flight /MALDI TOF" of them. Alpha-1-acid glycoprotein and clusterin were expressly down-regulated in breast cancer. (Cho, 2007)

3. Western blot: using this technique the expression of TRPM8 cells In breast cancer by estrogens regulation.

Western blot was used to resolve the expression of TRPM8 cells in breast cancer cells .

The calcium permeable cation is in excess expressed in cancer. The performance of the method was to examine appearance, purpose, and probable parameter of TRMP8 channels by estrogens receptor alpha cells in breast cancer.

TECHNIQUE INVOLVES , MCF -7 cell lyses in RIPA buffer and triton , sodium deoxycholate with human colon cancer tissue proteins extract in whole cell extract mixed using polytron homogenizer " PRO -200 , fisher bio block scientific ". Separation done by electrophoresis by SDS-PAGE and blot onto nitrocellulose membrane "(G E HEALTHCARE)", blot where carried out against antibodies which where compared with TRPM8, beta actin proteins. Blotting was done by chemiluminescence, bio rad.

RESULT:

The result obtain is TRMP8 which was articulated and serviceable in the breast cancer MCF -7 cell line. (Chodon et al., 2010)

3.Mass Spectrometry :

In proteomic analysis, it's the main method for biomarker discovery. method involves the direct evaluation of protein expression and also to recognize protein that are having degree of difference appearance between normal and tumour tissue , a range of cancers are detected include breast cancer. {{30 Wulfkuhle, Julia.D.Liotta, Lance.A.and Petricoin, Emanuel.F. 2003 ;}}

4.Comparison can be done between various techniques :

a. ELSA

b. Multidimensional protein identification

c. Proteomic pattern diagnostic

Methods

ELSA

Good application in the clinical assy.

Biomarkers recognition: biomarkers can recognise, but can be possible with mass spectrometry.

Multidimensional protein identification.

It has larger range of proteomes methods for the biomarkers recognition

Biomarkers recognition: yes

Proteomic pattern diagnostic

Its identifies the proteins, it goes along with diagnosis pattern analysis

Biomarkers recognition: it has highest recognition

5.Protein microarray

Protein micro array the combination technique shows the high throughput of identification of protein antibodies. Its gets combination with Mass Spectrometer it shows high throughput it has flexible format single analyse in large number of specimen. But required study prior to get the technique done .Deliberate by antibodies specificity and compassion need to be amplified prior to detection of tagged system. Protein microarray shows one of the most sensitive methods for discovery of biomarkers.

{{30 Wulfkuhle, Julia.D.Liotta, Lance.A.and Petricoin, Emanuel.F. 2003 ;}}

Methods

Practical day 1

Preparation of SDS PAGE GEL

Procedure:

1. Wash the two plates know as casting fame and pressure cams.

2. The plates overlapped on each other or fixed in such a condition that it should not have gap in between them.

Resolving Gel: preparation was done by adding following substance in following ml.

Acryl amide/bi-acryl amide 10.0 ml

1.5M Tris/hcl ph 8.8 5.0 ml

d H2O 4.8 ml

10% SDS 0.2 ml

Before adding this solution. Check for glass plate's leakage .Add this solution in tube before adding TEMED and APS. Because after adding both the solution gets solidified. As soon as the TEMED and APS are added in quantity of 0.01 and 0.1ml the whole solution is moved in the casting plate. Solution takes near about 15 min to get solidified

Stacking Gel:

Second solution

Above the marking line space is left for the filling of stacking gels It's the merging point of the both the gels. Preparation was done by adding following substance in following ml.

Acryl amide /bis-acrylamide 5.0 ml

0.5M tris/HCL ph 6.6 2.5ml

dH2O 2.4ml

SDS 0.1ml

Add the above substance in a tube, addition of TEMED and APS in quantity of 0.01and 0.5 ml.This causes the solution to get solidify. Necessary to add the solution in assemble above resolving gel. It again gets solidified in few minutes.

PRACTICAL DAY 2

Electrophoresis

After removing of comb, wells should be washed with running buffer. Be definite that gel cassette faces towards in the U shaped buffer tank. Add running buffer until half of the tank. Be alert about not overflowing the inner chamber.

Sample loading

Sample are being heated for 2 min at about 100 degrees temperature.sampes are loaded in the wells, be sure about not to prick to downward part of wells. Eventually settling at the bottom of the wells.

Four samples are used to load.

Marker, H2B histone proteins, H3.3 histone proteins, control sample, treated sample. Filled alternately in the each wells at about 20µl. Assemble the gel in buffer tank at 100˚C temperature for 35 min .Accurate heating of gel is necessary for the identification of the gel.

PRACTICAL DAY 3

Stain and De stain.

After removing the buffer tank by disconnect the power supply. Staining of gel is carried out. Separate the gel from the glass carefully with help of plastic gel releaser. It is necessary because gel may be break or loss. Cover the gel with paraffin plastic. Overnight staining is important.staning is done by Coomassie Blue. Remove the paraffin from the plastic box in which gel was present .Wash the stain from the gel, Wash the gel with the distilled water to remove the stain .It's now show desire bands on the gel.

Result

Wash the stains with water until bands are obtained.

Lastly, picture was recorded with the help of mobile camera.

RESULTS:

GEL IMAGE is obtained as follows

Result was not obtained.

Discussion:

Result was not obtained due to manual Errors

Errors.

Sample volume was excess

Improper proteolysis, the minimum time between sample preparation and electrophoresis

Poor polymerization takes place due to adding excess APS and TEMED.

Base of sample wells appears to be dragged downwards.

Protein bands are not seen properly due to improper loading of proteins into the gel.

Proteins bands are not recognised properly due change in the gel pore size.

Relevance of data

Experiment was carried out for the detection of estrogens cell in cancer with the help of two dimensional gel electrophoresis technique .it necessary of detection of cancer in prior stages. Experiment was done for identification of histone proteins such as H2B, H3.3, controlled and treated samples.

According to the following data, the experiment carried out shows the differential expression of estrogens stimulation.The main identification of histones H2A, A2B, H3 and H4. Method used in Identification of proteins by SDS- polyacrylamide gel electrophoresis with further analyses by mass spectrometry. Method involves detection of estrogens which are stimulating cells detection shows relative activity. Biomarkers are necessary to be relevant, as per identification of estrogens. Method was used for proper finding of biomarkers; it involves effect of different estrogenic compounds on ER receptors for protein expression. Result shows responsive proteins in MCF-7 cells were identified as histones.

{{23 Zhu, Zheying 2009 ;}}

Further experiment to validate the biomarkers.

As follows:

1. Mass spectrometry

2. LCM Laser capture Micro dissection.

3. Micro Array analysis.

4. ELISA (Enzyme linked immunosorbent assay)

Mass spectrometry

Mass spectrometry, the identification technique in protein-protein interactions. It is one of the most influential technology for recognize thousands of proteins. It is simple, shows exceptional mass correctness, soaring decision and sensitivity. It is one of the methods of choice for analyses of complex protein samples. Has strong application in genomic encoding and protein profiling. Mass can be used in combination with one and two dimensional method for more complex protein separation.

{{29 Aebersold, Ruedi 2003 ;}}

Laser Capture Micro dissection.

This technique provides opportunity to generate genes expression for the significant character genes. Detection of gene expression between normal and tumour cell in breast cancer cells this technique goes along with microarray for identification of different genes type in tumours.

{{24 Turashvili, Gulisa 2007 ;}}

Microarray Analysis.

It's a dominant technology in turn of phrase of thousands types of genes combination with LCM. Microarray Analysis shows different application such as tumour classification, molecular pathway modelling, functional genomics, and comparison of genes expression profiling.Affymetric array a Micro Array technique used in the breast carcinoma for genes differential expression. shown recognition between normal ductal and lobular cells PCR ( polymerase chain reaction validation of micro array result ) validation of micro array results in identification of collagen triple helix repeat containing 1( CTHRC 1) called as candidate gene was found in comparison of normal cells . It was reported in human solid tumours include cancer such as breast, thyroid, ovarian, cervix, liver and pancreas cancer progression. {{24 Turashvili, Gulisa 2007 ;}}

ELISA (enzyme linked immunosorbent assay).

Enzyme linked immunosorbent assay is Protein based assay. Cancer cells show the specific change in the genes expression. It is the most realisable technique for identification of such genes expression. This technique is reliable, sensitive, and widely available for detection of disease with high sensitivity. It is the protein based platform for detection and monitoring of cancer.

{{30 Wulfkuhle, Julia.D.Liotta, Lance.A.and Petricoin, Emanuel.F. 2003 ;}}