This video describes a procedure used to induce toxin synesis in ome species in this case for and the protocol for whole prote extraction used for proteomic studies in our laboratory. The length of the procedure, 16 days, four days for fungal growth, 10 days for toxin synesis, and two days for protein extraction. After four days, colonies are still actively growing toward the edge of the plate.
Typical aerial mycelium is formed and is collected using a steroid blade under lamina flow box by gentle scratching the surface of the plate. Mycelium is cut into five small pieces and added to an early my flask containing 25 milliliter of toxin inducing media. Toxin induction is more efficient than dark dust.
The flask is covered in aluminum. Cultures are then shaken for 10 days, 150 rounds per minute at 25 centigrade. The medium is of SAPH and contains high concentrated S row solution.
Bed together with darkness is known to facilitate toxins synesis in ria. The mycelium is then separated from the toxin containing liquid by filtering on the MI close tissue. The liquid can be used to measure toxin content while the mycelium is used for protein extraction.
To avoid media carryover, my ceiling is washed three times with sterile water. The excess water should be eliminated Afterwards, like liquid nitrogen is added, and samples can be stored at minus 80 or used for protein extraction. The protein extraction procedure is based on the use of SDS and TCA and consists of different lysis steps.
He will propose a method that compasses multiple operators to carry out. At the same time the extraction of biological replicates, a different operator carries out each replicate. This procedure takes into consideration differences in the extraction procedure that can be generated by different operators who are processing the replicates.
It should therefore show higher robustness when biological replicates are compared as the operator variable is included in the replicate. At the same time, the use of multiple operators reduces the time of processing. The sample is ground in a sterile mortar using liquid nitrogen until mycelium is a fine powder.
This step is crucial for the quality and amount of proteins. Mycelium is collected in 10 milli Teflon tubes in a proportion of four tenths of the total volume of the tubes. Lysis buffer containing SDS and different protease inhibitors is then added.
The tubes are then shaken until a homogeneous solution is obtained. Then the samples are shaken for 30 minutes in the code chamber to be further homogenized samples are boiled in order to dissolve cell walls and hydrophobic proteins. The presence of SDS prevents the formation of oligomers that abort precipitation of proteins.
A centrifugation step separates three phases. Proteins are suspended in the transparent solution that is collected in a new tube kept on ice. The palette is then used for performing a second ly step, repeating the vortexing, boiling and centrifugation.
The snat from the two lysis are combined in order to perform precipitation with glacial precipitation, buffer containing acid tone, tri acidic acid, and DTT. The samples are then stored overnight for 16 hours at minus 20 centigrade to allow protein. Precipitation proteins are situated at the bottom of the tubes to improve precipitation.
Tubes are centrifuge at high speed for 45 minutes. The palate is now well visible. Supernatants can be discarded using a five milliliter pipe pad.
TCA is then removed by adding 25 milliliter of glacial washing buffer containing acetone and DTT. It is then thoroughly and vigorously shaken. Then a new centrifugation step is performed.
The protein palate is now floating, so a tension should be paid to eliminate the snat. A second washing step is performed, adding washing, buffer, shaking and centrifuging. The SUP agent is eliminated and the sample is dried using a speed bag.
When the sample is dry, the protein is of powder like consistency. To store. The sample protein should be collected from the tube to diminish the electrostatic interference of plastic.
An electrostatic pistol is used and the protein powder is collected using a metal spatula. The proteins are then resus suspended directly in the labeling buffer. Used for 2D dash 30 minutes at 800 grams per minute.
Suffice for solubilizing. The protein before proceeding to the expensive 2D dash labeling. Protein quality is tested on SDS page one dimension gel as can be seen.
The absent of significant smears on the edge of a lane suggests a good quality of the sample.
