SDS PAGE technique

The SDS-PAGE technique is a fast, reproducible and low-cost method for proteins studies. This method is commonly used to investigate the purification steps, calculate the relative amount, and determine the molecular weight of proteins. This technique uses sodium dodecyl sulfate (SDS), which binds to proteins to cover their natural charge and creates a uniform distribution of negative charges on it, which binds to the hydrophobic regions of proteins and denatures them. Since many industrial projects are being carried out in Histogenotech Company and in each research project accuracy of study should confirm, it is necessary to carefully examine the proteins. This technique is able to accurately separate proteins based on their molecular weight. Polyacrylamide gel has very effective role in the separation of proteins in SDS-PAGE. The pore diameter of the polyacrylamide gel determines the separable weight range in SDS-PAGE. In this way, with different concentrations of gel, various proteins can be separated from each other.

Western blotting technique

Western blot is a laboratory method for distinguishing specific protein molecules from a mixture of proteins that is widely used in cell-molecular biology and immunology. This protein mixture can contain all the proteins associated with a particular tissue or cell. Numerous preclinical studies can be performed in the protein analysis laboratory of Histogenotech Company due to the existence of a diverse antibody storage that corresponds to different species of animals, including mice, rats, rabbits and humans. In these studies, the size of the desired protein can be evaluated using the Western blotting technique and the amount of protein expression can be measured. Western blotting is widely used in biochemical studies to qualitatively identify individual proteins and modify proteins (such as post-translational changes). This technique is commonly used to confirm protein production after cloning as well as in medical diagnosis.

Labeling the cell with GFP vector

One of the most novel methods for investigating the expression of recombinant proteins is the use of reporter genes in vector design. These vectors can be used to track the expression of the gene in cell, bacterial or yeast culture. Vectors that carry the green fluorescent protein (GFP) tracer gene are often used as reporter genes to report the expression of genes cloned in the vector or to track stem cells in vivo. In the biotechnology laboratory of Histogenotech, using lipofectamine, eukaryotic cells of different normal and cancerous categories were transduced with vectors containing GFP in doctoral and master’s thesis in different fields of basic medical or interdisciplinary sciences and after examination at different times. After infecting the cells with the virus, the expression level of GFP and the number of positive expression cells, counting and the efficiency of transfection and transduction methods are evaluated. GFP protein expression in transgenic animals can also be assessed in subsequent generations of modeled animal cells.

Perform PCR cloning on expressive vectors

After plasmid extraction, clonal PCR method is used to confirm the presence of the gene in the plasmid. Colony PCR is a technique used to examine the presence of DNA in plasmids inserted into bacteria and yeasts by the transformation reaction. The PCR cloning technique is performed directly on bacterial or yeast colonies, during which the target sequences are amplified directly by PCR without the need for DNA extraction and purification prior to PCR. Experienced specialized staffs in the molecular department of Histogenotech Company, in order to advance research projects in support of student projects, provide free consulting services to design suitable primers for PCR colonization. The primers are designed and synthesized before PCR cloning, and finally the PCR products are electrophoresed on agarose gel to observe the desired band. If the gene is in the right place, observing the band on the gel electrophoresis indicates that the colon selection process in the medium containing the appropriate antibiotic has been performed correctly.

Cell transduction with virus

Transduction is the process by which bacterial DNA is transmitted from one bacterium to another through a virus (bacteriophage). Generalized transduction is performed by lethal phages, and bacteriophage carries only bacterial DNA. So in this case the recipient cell will survive. After phage infiltrate the host bacterium, fragments of bacterial DNA remain intact and are randomly inserted into the phage capsid head. Phage will inject a bacterial DNA fragment it carries randomly into another bacterium. If there is a homology between the newly injected strand and the bacterial genome of the receptor, the fragment may be attached to it. The gene on that fragment can encode a protein that the receptor did not initially have, such as a protein that inactivates an antibiotic. The transferred gene fragment is protected from destruction by translocation by the phage capsid that holds it in place. Specialized transduction occurs by moderate phages (lambda phages). When the virus is activated, there is sometimes an error in cutting phage DNA, and a piece of bacterial DNA on one side of the phage is cut, cloned, and packaged with phage DNA. This action can lead to the transfer of that piece of bacterial DNA to other bacteria, and eventually it can give the bacterium a new capability.

Bacterial colony culture, plasmid purification, virus generation from vector

After the transformation, the transformed bacteria are cultured in liquid medium. Biomarkers are used to select bacterial cells that have received the plasmid. Biomarkers are usually a fluorescent protein or an antibiotic resistance gene. In order to evaluate the ability to produce a specific protein from a cell in clinical and preclinical studies, the use of expression vectors is very valuable. Plasmid purification in biotechnology laboratory Histogenotech is provide by selecting the bacteria carrying the plasmid, which is provide by culturing the bacteria in a solid culture medium containing a selective marker (antibiotic). In the molecular unit of Histogenotech biotechnology laboratory, plasmid purification is performed with a special commercial kit according to the manufacturer’s instructions. Methods for confirmation of recombinant plasmid in transformed bacteria include enzymatic digestion using restriction enzymes, sequencing and PCR colony using specific primers. Finally, for the initial confirmation of the presence of the desired gene in the plasmid, enzymatic cleavage is performed using the restriction enzymes and the presence of the expected fragments on the agarose gel will be confirmed by electrophoresis. Due to equipped microbial biotechnology laboratory of Histogenotech with TOP10 and DH5 E. coli host strains and designing and manufacturing bacterial-yeast expression vectors as well as plasmid extraction kits, biotechnology projects can be performed with the highest quality.

Bacterial Transformation by chemical method and electroporation

Transformation is a key step in the DNA cloning process. Different vectors are used depending on our purpose of the transformation as well as the size of the part to be transformed. Bacteria normally cannot receive extra-genomic DNA on their own. In general, two chemical and electrical methods are used to transfer the vector carrying the genome into the bacterium. The first method is to transfer the vector chemically. In this method, which is performed in microbial laboratory of Histogenotech, calcium chloride is used to create competitive bacteria. In this way, the bacteria are first placed in a cold solution containing calcium ions, which leads to the neutralization of the electrostatic repulsion between the negative charges of the membrane phospholipids and the negative charges of the vector DNA, and finally facilitates the process of vector transfer to the cell. In the second method, an electric field is used to increase the permeability of the bacterial wall (electroporation), which is done in Histogenotech Company.