#immuno

july 12th, 2021; 11:40 am.

back to business & writing some notes about immunology/biology🦠💉

i didn’t have a very aesthetics notes in this period of my life, but in my opinion on the second pics this drawing is too cute :(( it’s a small COVID virus with a white flag, because it’s simply gave up fighting with the world:D

Individuals with Paroxysmal Nocturnal Hemoglobinuria (PNH) have an acquired mutation in the PIG-A gene in a line of progenitor stem cells in their bone marrow. PIG-A normally codes for an enzyme that helps the glycophosphatidylinositol (GPI) protein anchor other proteins on the cell surface. The end result is that blood cells don't express a number of different proteins, including CD55 and CD59. CD55/59 deactivate the complement that occasionally binds to these "self" cells rather than invaders. Without those CD proteins, RBCs, WBCs, and platelets are susceptible to complement-mediated lysis. And get this: because most people breathe less deeply when they sleep, carbon dioxide increases slightly in their blood. CO2 is an acid, and complement-mediated destruction occurs more readily if there is a lower pH. The contents of all those broken cells are filtered out, and  in the morning the person may have "bloody" urine that is rust-colored from the heme (hemaglobinuria, not hematuria).

A western blot can detect specific protein molecules from a mixture of proteins.  They can also be used to evaluate the size of a protein of interest, and to measure the amount of protein expression. 

Gel electrophoresis

The proteins of the sample are separated by isoelectric point (pI), molecular weight, electric charge, or a combination of these factors. 

Samples are often boiled first to denature the proteins -  prevents proteases (enzymes that break down proteins) from degrading samples.

Most commonly, this uses polyacrylamide gels and buffers loaded with sodium dodecyl sulfate (SDS). SDS-PAGE (SDS polyacrylamide gel electrophoresis).

Proteins are covered in the negatively charged SDS - becoming anionic - and migrate towards the positively charged (higher voltage) anode through the acrylamide mesh of the gel. 

Smaller proteins migrate faster through this mesh, and the proteins are thus separated according to size.

The different rates of advancement (different electrophoretic mobilities) separate into bands within each lane. 

The concentration of acrylamide determines the resolution of the gel.

One lane is a marker/ladder - a mixture of proteins of known molecular weights, to act as a control and compare results to so as to estimate the sample’s molecular weight.

Transfer

To make the proteins accessible to antibody detection, they are moved onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF). 

Electroblotting uses an electric current to pull the negatively charged proteins from within the gel onto the membrane while maintaining the organization they had within the gel. 

Blocking

Blocking of non-specific binding prevents interactions between the membrane and the antibody used for detection of the target protein. 

Membrane is placed in a dilute solution of protein – typically 3–5% bovine serum albumin (BSA) or non-fat dry milk in tris-buffered saline (TBS) or I-Block, with a minute percentage (0.1%) of detergent 

The protein in the dilute solution attaches to the membrane in all places where the target proteins have not attached. 

When the antibody is added, it cannot bind to the membrane, and therefore the only available binding site is the specific target protein. 

This reduces background - clearer results, and eliminates false positives.

Primary antibody

A solution of primary antibody (diluted in either PBS or TBST wash buffer) is incubated with the membrane under gentle agitation. 

This binds to the target protein.

The membrane is washed several times in wash buffer to remove unbound primary antibody,

Secondary antibody

Membrane is exposed to another antibody 

Which recognises and binds to the species-specific portion of the primary antibody (eg anti-mouse secondary antibody will bind to almost any mouse-sourced primary antibody).

Secondary antibody is commonly linked to biotin or a reporter enzyme 

Several secondary antibodies will bind to one primary antibody and enhance the signal.

Western blot binding

As with most immuno tests, an enzyme can be attached to the secondary antibody that will produce a coloured reaction product - visible on the membrane so that the test can be read. 

Another method uses a near-infrared (NIR) fluorophore-linked antibody - light produced from the excitation of a fluorescent dye is static, making fluorescent detection more precise and accurate 

A radioactive label could also be coupled to the secondary antibody.

19.5.2020 — 23 / 100 days of productivity

pictured: immunology lecture

Anyway, I was reading through it, studying as one does when they are in college and I realized it sounds like one of them alien posts. Like humans find aliens and the over-excited alien is writing about the human post. Because this entire thing is like “the human immune system is highly complex! The innate immune system exists because the human’s ancestors had lived through any infection they had until the reproduced...Humans are exposed to many thing, including their own cells that may trigger reactions! Why that ever evolved...I have no idea.” That’s what this entire thing sounds like, complete with explanation points, my cue to know it is important information, color coded and reads like a guide.