What are the tips for using nitrocellulose?

Cellulose nitrate is a white polymer. This substance actually has many names, such as cellulose nitrate, and its English abbreviation is NC. This chemical substance has many characteristics, such as it easily changes color after being exposed to sunlight and it is also easy to burn. Therefore, cellulose nitrate is actually a dangerous substance. You must be cautious when using it. The following will introduce you to the correct use of cellulose nitrate.

Application tips for nitrocellulose membrane:

1. Principle of protein-membrane binding

The binding principle between protein and membrane, known binding forces include hydrophobic force, H bond electrostatic force, etc. The exact binding principle is not clear and is mainly supported by hypotheses. There are two main hypotheses:

1) First, the two are combined by electrostatic resistance, and then rely on H bonds and hydrophobic effects to maintain long-term combination.

2) First, the two are combined by hydrophobic interaction, and then the electrostatic effect is used to maintain the long-term combination.

Both hypotheses indicate that the binding process consists of two steps: first binding and then long-term binding. Due to the ambiguity of the binding principle, work in this area relies heavily on practical experience.

2. Effect of membrane on binding

1) Membrane pore size

Some technicians tend to use membrane pore size to distinguish different membranes, but please note that this is limited to products from the same manufacturer. If they are products from different manufacturers, this comparison is meaningless. The relationship between membrane pore size and chromatography speed has been described above.

As the membrane pore size decreases, the actual available surface area of ​​the membrane increases, and the amount of membrane-bound protein also increases. The parameter for estimating the surface area is the surface area ratio (the ratio of the actual available surface area to the membrane surface area used).

In addition, the smaller the membrane pore size, the slower the chromatography speed, the longer the time it takes for the gold-labeled complex to pass through the T line, and the more complete the reaction.

Combining the above two points, the conclusion is that the smaller the membrane pore size, the higher the sensitivity. But at the same time, it also slows down the plate running speed and increases the chance of nonspecific binding, that is, the higher the false positive. Therefore, it is necessary to select the membrane suitable for the actual project according to the test results and find the right balance.

2) Differences in membranes from different manufacturers

This difference mainly comes from two points:

1> When producing membranes, the sources, types, and quantities of polymers and surfactants used are different. Similarly, these two types of substances generally have a greater impact on performance in membrane processing.

2>The processing process is different.

3. Biological raw materials, reagents and formulations of buffer solutions

1) Biological raw materials. The use of biological raw materials for CT lines varies, so only a brief description is given here.

First, monoclonal antibodies bind better to membranes than polyclonal antibodies, mainly because polyclonal antibodies have many different surface sites, and the optimal binding conditions for each site to the membrane are slightly different, which undoubtedly increases the difficulty of optimization.

Secondly, the larger the molecular weight, the more difficult it is for the protein to bind to the solid phase material.

2) Buffer

What everyone is most concerned about is probably the desire to obtain a formula with excellent performance, including buffer, blocking solution and other processing solution formulas.

In fact, it is impossible to provide a universal formula list. Because different reaction systems require different formulas to support, and the reaction systems of different institutions are different. If you want to catch "fish", you must first learn "fishing". In order not to mislead everyone, the following questions involving formulas only provide ideas, and please explore the specific formula yourself.

The composition of buffer is generally: PBS (or other buffer system) + action substance (for a specific problem) + PH adjustment. After referring to various previous materials, my personal opinion is that the formula principle should be simple rather than complicated, and the action substance should be added according to your needs. Many of the original action substances that need to be added are no longer needed due to the improvement of membrane manufacturing technology.

The recommended buffer system is 0.01M PBS pH 7-7.2, which has good adaptability to a variety of antigens and antibodies.

The following are the main factors that affect the performance of the substance:

A small amount of NACL can reduce signal strength and eliminate false positives.

Organic alcohol (methanol, isopropanol, etc.) wets the membrane, reduces static electricity on the membrane, and facilitates the binding of the coating. I personally do not recommend it because the membrane making process has been improved.

Surfactants (TW20, TX100) can increase hydrophilicity, avoid hollow lines, and enhance color.

Sugar, a protective agent, slows down the aging process and can also increase hydrophilicity as above.

Adjusting the pH to a certain position can eliminate false positives.

4. Sample environment

Ambient humidity is very important for the film deposition process. The optimal humidity is generally between 45-65%.

When the humidity is too low, static charge is easily accumulated on the membrane, and scattered points are easily formed on the membrane, resulting in hydrophobic spots in the test.

When the humidity is too high, the capillary action on the membrane is strengthened, and the film is prone to cause the CT line to become wider or even diffuse.

In order to ensure the uniformity of the membrane humidity during sample spotting, the membrane is usually placed under the humidity condition for a period of time before sample spotting.

5. Relationship between spotting instrument and membrane surface condition

There are currently two ways of spotting, the scratching film type and the non-contact spotting film type. The non-contact spotting film type is better than the scratching film type, and the imported scratching film type is better than the domestic scratching film type.

Since the membrane-scratching method uses a soft tube to draw the antibody onto the membrane surface, and the membrane itself is soft and brittle, the scratching tube will leave marks on its surface. Imported membrane-scratching machines use better materials and control systems, so the scratches left are lighter, while domestic instruments are inferior, so the scratches left are more serious. Scratches can easily form resistance to the gold-labeled complex of the chromatography, resulting in false positives. At the same time, it is easy to have a strange phenomenon that a thin line (ghost line) appears at the T-line position when running the plate, and the ghost line disappears after the running plate is finished.

6. Membrane width and spotting position

The width of the membrane is generally 18mm (or 20mm) and 25mm, which are used for test strips and test plates respectively.

However, different T-line spotting positions will bring different sensitivities. When the spotting position moves upward, the speed of the gold-labeled complex passing through the T-line position slows down, the reaction time increases, and the sensitivity increases. Conversely, the sensitivity decreases. This method can be used to change the sensitivity and eliminate false positives.