Hawach To Talk About The Applications And Differences Filter Membrane
1. Nylon membrane
Nylon membrane is a synthetic long-chain polyamide membrane, which has a strong binding ability to nucleic acid and protein and can replace nitrocellulose membrane for molecular imprinting and hybridization experiments.
Nylon membrane features good temperature resistance, which can withstand 121℃ saturated steam hot-press sterilization for 30min, maximum working temperature is 60℃; also, it has good chemical stability, which can withstand dilute acid, dilute alkali, various organic and inorganic compounds such as alcohols, esters, oils, hydrocarbons, halogenated hydrocarbons, and organic oxides. It has the widest applications, such as aqueous solution and organic mobile phase filtration; filtration of beverage and chemical liquid; filtration of tissue culture medium, and so on.
2. PVDF-Polyvinylidene fluoride membrane
PVDF membrane, or polyvinylidene fluoride membrane, is a kind of solid-phase support commonly used in Western blotting. The PVDF membrane is hydrophobic, and the pore size of the membrane can be large or small. As the pore size of the membrane continues to decrease, the membrane is more firmly bound to low molecular weight proteins. For proteins larger than 20000, a 0.45um membrane is used, and for proteins smaller than 20000, a 0.2um membrane is used. The use of the PVDF membrane requires pretreatment. The purpose of methanol treatment is to activate the positively charged groups on the membrane, making it easier to bind to negatively charged proteins. With high mechanical strength, the PVDF membrane is an ideal solid support material in the imprinting method.
3. NC membrane
Nitrocellulose filter membrane (NC membrane for short) is used in Northern Blot, Southern Blot, and Western Blot. Hybridization techniques include solid-phase hybridization and liquid-phase hybridization. Solid-phase hybridization technology is currently more commonly used. First, the nucleic acid to be tested is bound to certain solid-phase support and then hybridized with the labeled probe in the liquid phase. Nitrocellulose membranes are commonly used for solid supports.
Comparison among the above three
Nylon membrane is an ideal nucleic acid solid support and there are many types; nitrocellulose membrane is currently the most widely used solid support with the cheapest price; the PVDF membrane is somewhere in between.
1. In terms of binding capacity: nylon membranes can bind DNA and RNA up to 480-600μg / cm2, and can bind nucleic acid fragments as short as 10bp; nitrocellulose membranes can bind DNA and RNA up to 80-100μg / cm2. The 200bp nucleic acid fragment has a weak binding ability; the PVDF membrane can bind DNA and RNA up to 125-300μg / cm2.
2. In terms of temperature adaptability: after the nylon membrane is baked or irradiated with ultraviolet rays, some of the pyrimidine bases in the nucleic acid can be combined with the positive charge on the membrane; the nitrocellulose membrane relies on hydrophobic interaction to bind DNA and the binding is not strong; The PVDF membrane is firmly bonded and resistant to high temperatures, and is particularly suitable for Western blotting.
3. In terms of toughness: the nylon membrane is stronger; the nitrocellulose membrane is more brittle and easily broken; the PVDF membrane is stronger.
4. In terms of repeatability:
(1)The nylon membrane can be used repeatedly for molecular hybridization. After hybridization, the probe molecules can be eluted by alkali denaturation; the nitrocellulose membrane cannot be reused; the PVDF membrane can be reused.
(2) The use of the NC membrane is also very simple. For example, no formaldehyde pretreatment is required, as long as it is infiltrated on the surface of non-ionized water to expel the air bubbles in the membrane, and then equilibrated in electrophoresis buffer for a few minutes; the NC membrane is easy to close, nor particularly strict cleaning conditions are required. The protein transferred to the NC membrane can be stored stably for a long time under appropriate conditions, but it should be noted that the pure nitrocellulose membrane is relatively brittle and easy to roll, and the operation should be careful during use.
Because it can’t stand many “tortures”. When choosing a nitrocellulose membrane, it is necessary to choose a suitable pore size. Generally, large molecular proteins above 20KD use a membrane with a pore size of 0.45um. If the membrane is less than 20KD, it is recommended to choose 0.2um. Also, note that the selection of pure NC membrane mixed with cellulose acetate (CM) -containing the NC membrane will reduce the binding force.
(3) Since the protein-bound on the NC membrane will be replaced by some detergents, use a milder Tween20 when blocking, and the concentration should not exceed 0.3%. Generally speaking, the purer the NC membrane, the higher its protein binding capacity, so it is a consideration to increase the sensitivity and resolution of WB and improve the purity of the membrane used. If the NC membrane is mixed with some cellulose acetate, which has been mentioned before, it will affect the protein binding
(4) Compared with nitrocellulose membranes, PVDF membranes have superior performance in protein retention, mechanical strength, and chemical compatibility. The typical binding capacity of commercially available nitrocellulose membrane is 80-100μg / cm2, while the binding capacity of the PVDF membrane is 100-200μg / cm2 (and the binding strength PVDF is 6 times stronger than nitrocellulose membrane!). But the biggest advantage of PVDF membranes is not only this: better mechanical strength and chemical resistance make PVDF membranes ideal for various staining applications and multiple immunoassays, and a single lane copy of the gel can be used for multiple purposes, in particular, it is necessary to do N-terminal protein sequencing.
Under fairly “severe” cleaning conditions, the PVDF membrane remains intact when the nylon or nitrocellulose membrane has been degraded, so PVDF is also the best choice for protein sequencing. But it is not suitable for fluorescence. Particular attention should be paid to PVDF membranes that require 100% methanol pretreatment (not more than 15 seconds) and then equilibrated with buffer before they can be used.
Hawach is specializing in providing different kinds of filter membranes to meet various lab requirements. Welcome to visit hawach.com for more details.