WHAT TO KNOW ABOUT PROTEIN LYSATE FROM TISSUE
Lysis is simply the breakdown of cell membrane via enzymatic, viral or osmotic mechanism. Lysate is the fluid that contains the lysed cell contents. Lysis is used to analyze and determine the composition of nucleic acids, lipids and proteins (as complexes or individual compounds), as it applies to western and southern blotting. Protein purification, DNA and RNA extractions or purification of organelles is also made possible by lysis. Simply put that protein lysate is the protein fluid prepared from the lysed tissue samples, defected or not. Protein lysate can also be prepared from cell culture.
SOURCES OF PROTEIN LYSATE FROM TISSUE
Protein lysate from tissue are obtained from isolated tissues of test subjects (especially animals). The tissues, however, may be defected or non-defected, depending what the protein lysate is to be used to determine or used for.
PREPARATION OF PROTEIN LYSATE FROM TISSUE
• The tissue sample collected is dissected on ice quickly, to avoid degradation by proteases.
• Then placed in suitable micro-centrifuge tubes and then immersed in liquid nitrogen to snap freeze. Samples are then stored at about – 800C. They can be used afterwards or kept in ice for instant homogeneity.
• A 300 μL of ice cold lysis buffer is added to a 5 mg of a piece of tissue rapidly to the tube. An electric homogenizer is used to homogenize it and the blade is rinse twice each with same volume of lysis buffer.
• Then, let the agitation be maintained for 2 hours at about 40C. Based on the amount of tissue present is how you should determine the volumes of lysis buffer.
• To avoid loss of protein and large volumes of samples to be loaded onto gels, protein extracts should not be too diluted. Recommended minimum concentration is 0.1 mg/mL, while the optimized concentration is 1-5 mg/mL.
• Subject to centrifugation for 20mins at 12,000rpm and at 4oC still inside a micro-centrifuge. Remove the tubes gently from the centrifuge and place on ice. Let the supernatant be aspirated and the place in a fresh tube to remain on ice.
USES OF PROTEIN LYSATE FROM TISSUE
They have vast application in molecular biology, biochemistry, bio-engineering and laboratory works that are involved in cell biology researches. Their uses in these areas are as follows:
• Components of cell cultures can be subjected to lysis with the aim of isolating and purifying its component – purification of protein, purification of cell organelles and extraction of DNA and RNA.
• Specifically, protein lysate obtained from tissue can be analyzed to determine the measure, type and composition of the proteins present and how they can cause tissue specificity, considering their DNA and RNA related functions. It is good you know that there are tissue-specific proteins.
OTHER INFORMATION ABOUT PROTEIN LYSATE FROM TISSUE
Protein lysate generally are handle with utmost care and preserved according to the necessary conditions for the temperature (about -800C) and storage material. This is because proteins are easily denatured or degraded when subjected to any unfavorable condition or when it comes in contact with contaminants like detergents, enzymes or pathogens.
Also, there is a maximum shelf-life for protein lysate from tissue: some can still stay well for six months if properly preserved in the necessary storage conditions. However, protease inhibitor can be added to some, to prolong the shelf-life.