CD markers and Cancer

CD is short for Cluster of Differentiation.
The CD system is commonly used as cell markers in immunophenotyping, allowing cells to be defined based on what molecules are present on their surface. All cells in our body have one or more of them and they are useful for classifying white blood cells / WBC. There are more than 250 types of CD molecules. 

CD and their connection with cancer diagnosis

The most precise way to identify the different types of WBC is to see what CD molecules appear on their surface. CD markers are mostly useful for classifying WBC and especially important for diagnosis of lymphomas and leukemias. To find out the type of WBC affected and the exact type of leukemia and lymphoma, we need to identify the types of CD molecules that the cancer cells have.

To give example, two lymphomas diffuse large B-cell lymphoma (DLBCL) and anaplastic large cell lymphoma (ALCL) looks similar under the microscope with large cancer cells, even though, they have different prognosis and treatments. Pathologists often use CD markers to distinguish between different lymphomas. DLBCL is CD20 positive, while ALCL is CD20 negative but positive for CD30. 

CD molecules role as cancer treatment

CD marker-specific antibodies have been widely used for cell sorting, phenotyping, and blood cancer diagnosis.  In addition, CD markers have become significantly important for cancer treatment. Some therapeutic antibody drugs have been designed to identify and attack cells that have a particular type of CD molecule. (rituximab to CD20 for lymphomas and leukemia treatment; alemtuzumab to CD52 for chronic lymphocytic leukemia and T-cell lymphoma treatment).

These drugs are called monoclonal antibodies and they can attack only the type of cell that contains the specific target CD molecule. Monoclonal antibodies can also be tagged to drugs or radiation-emitting substances that add to the ability to kill cells that have the specific CD marker on their surface.

Reference:

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Bradford Protein Assay

The Bradford assay is a protein determination method that involves the binding of Coomassie Brilliant Blue G-250 dye to proteins . The dye exists in three forms: anionic - blue,  cationic - red, and neutral - green.

In the acidic environment of the reagent, Under acidic conditions, the dye is predominantly in the doubly protonated red cationic form.  When protein binds to the Coomassie dye, it results in a spectral shift from the reddish/brown form of the dye to a stable un-protonated blue form. The blue dye form is measured using a spectrophotometer or microplate reader

Development of color in Bradford protein assays is associated with the presence of certain basic amino acids (primarily arginine, lysine and histidine) in the protein. The principal for protein binding between dye and Protein is described by Van der Waals forces and hydrophobic interactions. The number of Coomassie dye bound to each protein is approximately proportional to the number of positive charges found on the protein.

Free amino acids, peptides and low molecular weight proteins do not produce color with Coomassie dye reagents. Generally, the mass of a peptide or protein must be at least 3000 daltons to be detectable with this reagent.


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Choosing Protein Assay : BCA VS Bradford

Both Assays are biochemical assay used to determine the total concentration of protein in a solution

Bicinchoninic acid assay (BCA assay) is a Copper-based protein assays , which is also known as Smith assay. The total protein concentration is shown with a change in sample solution colour from green to purplish in proportion to protein concentration, which then measured using colorimetric techniques.

Some benefit compared to Bradford and Lowry assays is that BCA Protein Assay is compatible with samples that contain up to 5% surfactants / detergents. Also,  BCA Assay responds more uniformly to different proteins compared to Bradford method.

more info on BCA Assay

The Bradford assay, a colorimetric protein assay and dye-based Protein Assay Chemistries, is based on an absorbance shift of the dye Coomassie Brilliant Blue G-250 in which under acidic conditions the red form of the dye is converted into its bluer form to bind to the protein being assayed.

Compared to other assay, Bradford is fast and easy to perform since it is performed at room temperature and no special equipment is required. The This coomassie dyed based assays are compatible with most salts, solvents, buffers, thiols, reducing substances and metal chelating agents encountered in protein samples.

The main disadvantage of is their incompatibility with surfactants at concentrations routinely used to solubilize membrane proteins. The presence of a surfactant in the sample will causes precipitation of the reagent. Coomassie dye-based is highly acidic, therefore proteins with poor acid-solubility should be avoided. Finally, Bradford Assay result in about twice as much protein-to-protein variation as BCA Assay

more info on Bradford Assay

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PCR Basic

Finding best condition for optimal PCR result can be tedious and frustrating. Optimising PCR workflow include improvements in sample prep time, reaction time, reaction sensitivity, reproducibility and ease of use. 

If you often do PCR in the lab, a good way to save time to to create a master mix. PCR mix normally contains buffer, taq polymerase and deoxynucleotides. In the market, there are PCR mixes sold for simplicity. 

Some brands added perks such as dyes, PCR enhancing additives and designed DNA polymerase. Commercial PCR mix is quality controlled which can be important to avoid error in homebrew PCR master mix.

Chemicals and reagents that are part of PCR reactions are crucial for routine success. For PCR workflow, significant amount of time is spent to prepare the DNA. There are plenty PCR kits in the market that help to save time and optimised result. Most companies will designed unique PCR buffer recipe to work in a range of PCR reactions and templates. 

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Taq Polymerase

What is Taq Polymerase?

Taq DNA Polymerase is an thermostable DNA polymerase that catalyzes dNTPs into DNA, is frequently used in polymerase chain reaction (PCR). The name comes from thermophilic bacteriumThermus aquaticus

Why don't we basic Taq Polymerase?

Taq polymerase has many limitation such as low replication fidelity. They lack a 3' to 5' exonuclease proofreading activities with error rate 1/9,000. Standard Taq used in most lab still require time to optimise their PCR reaction, which can be tedious and time consuming. Therefore there are many modification made to improve basic Taq Polymerase for simplify, speed up and better performance. 

Ref: link1, link2, link3