#assay

In the development of small molecule drugs, it is necessary to evaluate the bioavailability of the compound as early as possible, so as to lay the foundation for subsequent development. In addition to using experimental animals to evaluate bioavailability, various in vitro analysis methods have been developed to evaluate the ability of compounds to penetrate the intestinal wall. These in vitro experiments are not only lower in cost but also higher in throughput and shorter experimental time. Among them, Caco-2 monolayer cell permeability analysis assay has become one of the standards in vitro methods.

CaCO-2 cells are derived from human colon cancer and have the typical characteristics of intestinal epithelial cells, such as the formation of a polarized monolayer columnar epithelium, and densely differentiated microvilli on the top surface of the cells neatly arranged to form a striated border and intercellular connection.

The determination of Caco-2 cell permeability not only helps to determine intestinal permeability but also can identify specific transporters or efflux proteins and intestinal phase II drug-metabolizing enzymes. Therefore, this Caco-2 permeability assay provides valuable information for drug development in an effective and reproducible manner, which is not available in non-biological models, and even in many other biological models.

The Efflux Ratio can be calculated by measuring the transport from A-B and B-A in two different directions, so as to determine whether a molecule has active efflux.

Caco-2 Permeability Assay Protocol

Compound required

10 mM DMSO 100µL or 2 mg

Compound Test Concentration

10µM

Cell Culture

21 days

Replicates

N=2

Incubation Conditions

Incubation in CO2 incubator at 37ºC for 90 min

Positive control compound

digoxin （or other compounds）

Integrity of the monolayer

TEER>200 Ω•cm2

Analysis method

LC-MS／MS

Data delivery

Papp, Efflux Ratio, %Recovery

Data calculation

Papp = CRecx VRec/ (A x t x C0) Efflux Ratio = Papp (B→A) /Papp (A→B) Recovery = [(Vrec x Crec) + (Vd x Cd)] / (Vd x C0)

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.

I explain it as “you don’t know anything about Chinese culture in general, read this assay, pretty please”: https://bjdrecastpositive.tumblr.com/post/172968722887/httpsaeoncoessayswhy-in-china

But I’ll answer each question individually for you: (since you seem to be struggling to understand it as a concept)

1 . “you say that oueniefs never claims to be the legit company. So how would you explain his sales pages now using the company name,”

First off, it’s not “now”, they have always used it. And of course they need a way for people to find a certain doll. They don’t own Volks as a company or a brand, and they haven’t sculpt X model, but, it needs to be written in the title of the sale and description, because it’s A RECAST OF X DOLL from X BRAND. This doesn't claims ownership, you will never read Oueneifs telling you “we sculpted the 4 sisters” or “we own Volks brand”, no.

2 . “a logo he copy righted,”

Except that “looks like” it’s not a legal issue. Disney font has been free to use and available for quite a while now. And even so, Disney it’s not a chinese company, and if Disney had so many issues (like you seem to have) with it, trust me, it’s DISNEY, they own MARVEL cinematic universe and STAR WARS, If they wanted to erase Oueneifs from the planet, they would. And yes, Oueneifs logo it’s copyrighted on China, because continental Chinese law allow it, thus, it’s not illegal in any way.

3 . “packaging meant to look legitimate,”

Noooou, the packaging on only SOME items sold at Oueneifs Store are meant to look NICE and presentable for a gift (it doesn’t even copy the original design of the legit box, anyone can tell, this aren’t “Soom” or “FL” boxes, this are STORE boxes, from the Oueneifs Store), in most cases dolls are shipped wrapped in plastic bubbles and a simple plain brown cardboard box, if you where REALLY someone who owns or buys recasts, you would KNOW. lol

4 . “fake COAs?”

They’re not meant to replace the legit ones. It’s not illegal to print a free copy of it since they don’t have the watermarks and it has no costumer and client and order number from the legit company, it’s just a nice cool piece of paper for you to keep. God knows I keep forgetting the exact date I brought my dolls, and I have to check the date I filled for my own sake on this papers.

5 . “I only buy the dolls to paint and resell because the legit ones are are stupid expensive but he very clearly looks like he's claiming to be this company or at least being a legit reseller for them. How can you deny that?”

I don’t deny it, never did. Oueneifs, for starter, never, EVER claimed to be “the company” that designed the original sculpt, just pointed out “what sculpt it was and what was the origin of it”, second, IT IS a LEGIT RESELLER STORE in China. Under (continental communist) Chinese law, the store Oueneifs, it’s copyrighted as a brand. You might be surprised by the concept, but some people reproduce recasts from Oueneifs due to their reputation and cheat buyers to buy a lesser quality reproduction of Oueneifs recasts. At the same price. This is bad for the buyer who will get a cheap recast thinking it has brought it from Oueneifs, and it bad for Oueneifs, that will get bad reviews for the lack of quality of another seller. This is why, Oueneifs as a STORE brand it’s copyrighted and belongs to ChinaBJD. Now, I sense you haven’t been around here for long, but, for instance, the company DollZone was selling cheap reproduction of Volks dolls on 2005, only on 2008 DollZone established their first legit sculpts. Look it up on this blog for more information if you like. THAT was “claiming DollZone owned Volks models”, and now DollZone seems to be a saint and no one remembers where it came from. So get your s*th in line, soldier.

Exploring Jewelry Definitions – Assay

Assay

A chemical spot test that can help identify the approximate composition of a gold or silver alloy.

A benchmark is used to obtain a very small sample of the metal through abrasion, which is subsequently exposed to different types of acids.

The disintegration of the streak on the benchmark by a given acid indicates the approximate chemical composition, for example, 14 karat versus…

la musica è forse il più bel regalo da fare ad un figlio.

Urea and creatinine assays