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Cell Lysis Method Protocol Stanica Lysis Method Protocol

Information on the cell lysis technique here. Informacije o cell lysis tehnika ovdje.

Cell Lysis Methods Table of Contents Stanica Lysis Metode Table of Contents

• What is Cell Lysis? Što je Stanica Lysis?
• Definition Definicija
• Background Pozadina
• Applications Aplikacije
• Limitations Ograničenja
• Factors to consider for Cellular Lysis Čimbenika da razmotre za staničnu Lysis
• Protocol Protokol
• Agarose Gel Electrophoresis Videos Agarose Gel elektroforeza Video
• Other Cell Lysis Protocols Ostalo Stanica Lysis Protocols
• Troubleshooting Cell Lysis UPOZORENJA Stanica Lysis
• Discussion Topics for Cell Lysis Teme za raspravu Stanica Lysis
• Get Help on your Cell Lysis Problem Zatraži pomoć na vašem Stanica Lysis Problem

What is Cell Lysis? Što je Stanica Lysis?

Cell lysis or cellular disruption is a cell biology method for the release of biological molecules including organelles, proteins, DNA, RNA and lipids from inside a cell. Cell lysis ili stanični poremećaj je stanična biologija način za oslobađanje od bioloških molekula, uključujući organelles, proteini, DNA, RNA i lipidi iz unutar stanica.

Background on Cell Lysis? Pozadina na Stanica Lysis?

For most cells, mild osmosis is usually enough to lyze the cells. Za većinu stanica, blage osmoza je obično dovoljno da lyze ćelije. This is done by lowering the ionic strength of the surrounding solution, causing the cells to swell and burst releasing their contents. To se radi tako smanjiti snagu ionic okolnih rješenje, uzrok stanice do oteklina i prasak izdati njihov sadržaj.

Mild surfactans are often used in addition to mechanical lysis methods. Blaga surfactans se često koristi u Pored mehaničkih lysis metode.

Mechanical or Physical Lysis Methods include: Mehanička ili fizička Lysis Metode uključuju:

• mechanical agitation mehaničko miješanje
• pressure pritisak
• sonication
• nitrogen bomb or nitrogen burst lysis method nitrogen bomba ili dušika praska lysis metoda
• ultrasound with small probes ultrazvuk s malom proba

These steps are conducted to ensure complete dissociate of the cell and its cellular components including breakdown of organellar and nuclear membranes. Ovi koraci su provedena kako bi se osiguralo potpunu rastati se od stanica i stanični njene komponente, uključujući kvar organellar i nuklearne membrane.

Due to the high costs of growing cells, usually only a small amount of cellular material is available. Zbog visokih troškova uzgoj stanica, obično samo malu količinu stanični materijal je dostupan. It is thus necessary to be efficient when lysing cells to ensure the maximum of usable material is obtained. To je tako potrebno da bude efikasna kada lysing stanice kako bi se osiguralo maksimalno iskoristiv materijal je dobiven. Often, several methods are used in combination to ensure almost complete lysis. Često je nekoliko metoda se koristi u kombinaciji kako bi se osiguralo gotovo kompletna lysis.

Applications of Cell Lysis Applications of Stanica Lysis

Cell lysis is vital for the extraction of DNA, RNA and Proteins from cells. Cell lysis je bitno za ekstrakciju DNA, RNA, Proteini iz ćelije. Therefore, it is needed in most cell biology techniques and even molecular biology techniques. Stoga je potreban da bi se u većini tehnike stanične biologije i molekularne biologije i tehnike.

Limitations of Cell Lysis Ograničenja Stanica Lysis

Cell lysis is limited in its usage as it requires the cell membrane to be pierced and cell contents to be released. Cell lysis je ograničena u korištenju kao što zahtijeva da se stanična membrana cell proboli i sadržaja da bi bilo objavljeno. These lysed cells die and cannot be cultured or grown again. Te stanice lysed umrijeti i ne može biti uzgojenom ili ponovno narasla.

Factors to Consider for Cell Lysis Čimbenika da razmotre Stanica za Lysis

There are many factors to consider which cell lysis method to use or how to conduct it. Postoje mnogi faktori koji razmotriti stanica lysis metoda korištenja ili kako ga provoditi.

Volume of Cells Volume of Cells

The amount of cells to lyse is an important parameter in cell lysis. Iznosu od stanice do lyse je važan parametar u cell lysis. If you only have a very Ako imate samo jedan vrlo

If only a few microliters of sample are available, care must be taken to minimize loss and to avoid cross-contamination. Ako samo nekoliko microliters uzorka su dostupni, zaštita mora biti poduzeti kako bi se umanjili gubici i da bi se izbjegla cross-kontaminacije.

Disruption of cells, when hundreds or even thousands of liters of material are being processed in a production environment, presents a different challenge. Poremećaj u stanice, kada stotine ili čak tisuće litara materijal se obrađivati u proizvodnji okoliša, predstavlja drugačiji izazov. Throughput, efficiency, and reproducibility are key factors. Propusna moć, učinkovitost, i reproducibilnost su ključni čimbenici.

Number of Cell Samples to Lyze Broj Stanica Uzorci za Lyze

Often there are many cell samples to lyze. Često ima mnogo stanica uzoraka na lyze. There are many issues to consider when doing this including cross contamination of samples, the speed of processing, and equipment cleaning after each sample is lysed. Postoje mnoga pitanja za razmatranje kada to uključujući križ kontaminacija uzoraka, brzinu obrade, i oprema za čišćenje nakon svakog uzorka je lysed.

Difficulty of Cells to Lyze Težine od ćelije do Lyze

Some cells are quite difficult to lyse. Neke stanice su prilično teško lyse. As the lysis difficulty increases, a greater lysis force or higher ionic strength of buffers is required to efficiently disrupt the cells. Kao lysis poteškoća povećava, veći lysis ili više ionic force snaga buffers je potrebno za efikasno ometalo stanice.

For more difficult lysis of cell samples such as yeast samples, there is a steep increase in the processing power, time and cost required. Za teže lysis of stanica uzoraka kao što je kvasac uzorci, nalazi se strmi porast u procesorskoj moći, vremena i troškova potrebnih.

For the most difficult samples to lyze, such as bacterial spores, these require mechanical forces combined with chemical or enzymatic efforts. Za najteže uzoraka na lyze, kao što su bakterijska spores, te zahtijevaju mehanička snaga u kombinaciji s kemijska ili enzimska napore. The problem is even with multiple and harsher methods, usually one achieves limited disruption efficiencies. Problem je čak i sa mnogostruk i harsher metode, obično jednom ograničenom poremećaj postiže djelotvornost.

Efficiency of Cell Lysis Učinkovitost Stanica Lysis

Depending on what part of the cell, organelle or fraction you need to isolate, over-lysis may affect your desired product. Ovisno o tome što je dio stanica, organelle ili frakcija vam je potrebno za izoliranje, nad-lysis svibanj utjecati na Vaš željeni proizvod.

If subcellular fractionation is used, it is more important to have a delicate lysis to achieve an intact subcellular organelle components. Ako subcellular fractionation se koristi, to je više važno imati delikatan lysis postići netaknuti subcellular organelle komponente. Obviously, you will achieve a lower efficiency of lysis and thus will require more cells. Očito, ti će postići nižu učinkovitost lysis, a time će zahtijevati više ćelija. To scale these cell lysis processesses, the time to disrupt the cells and the reproducibility of the method become more important factors. Da razmjera tih stanica lysis processesses, vrijeme bi ometalo stanica i reproducibilnost od metoda postati važniji čimbenici.

Cell Lysis vs Molecule to be Isolated Stanica Lysis vs Molekula da se osami

It is very important to cater the lysis method to the protein you need isolated. Vrlo je važno da se razonoditi u lysis način da vam je potrebno proteina izolirane. If it is a nuclear protein, care is required to lyse the cell first and then isolate the nuclear membrane. Ako je to nuclear protein, njega je potrebno za lyse je prvi stanica, a zatim odvojiti od nuklearne membrane. After the nuclear membrane is isolated, then one may lyse the nuclear membrane and release the molecule of interest. Nakon nuklearne membrane je izoliran, zatim jedan svibanj lyse nuklearnu membranu i objave molekula od interesa. This methodology decreases the contamination of molecules that are from other cellular compartments/organelles. Ova metodologija smanjuje onečišćenje od molekula koje su od drugih stanični compartments / organelles.

Furthermore, depending on the molecule care must be taken to use or not use certain lysis solutions or methods. Nadalje, ovisno o molecule skrb moraju biti poduzeti kako bi se koristiti ili ne koristiti stanovite lysis rješenja ili metode. For example, if you are trying to isolate a phospho-protein that is sensitive to phosphatases, it is not a good idea to lyse and expose the phospho-protein to thousands of proteases and phosphatase enzymes. Na primjer, ako pokušavate izolirati phospho-jedan protein koji je osjetljivo na phosphatases, to nije dobra ideja da lyse i otkriti phospho-protein tisućama proteases fosfataza i enzimi. Protecting your molecule from harsh or enzymatic conditions is important. Zaštita vaše molekule s otežanim uvjetima ili enzimska je važno. The temperature (low temperature of lysis), and usage of inhibitory molecules (such as protease and phosphatase inhibitors) is important in these lysis cases. Temperatura (Niska temperatura od lysis), i korištenje inhibitory molecules (kao što su protease i fosfataza inhibitori) je važno u tim slučajevima lysis.