Processing of Samples
Serum - Use a serum separator tube and allow samples to clot for two hours at room temperature or overnight at 4oC before centrifugation for 20 minutes at approximately 1000×g. Assay freshly prepared serum immediately or store samples in aliquot at -20oC or -80oC for later use. Avoid repeated freeze/thaw cycles.
Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000×g at 2 - 8oC within 30 minutes of collection. Remove plasma and assay immediately or store samples in aliquot at -20oC or -80oC for later use. Avoid repeated freeze/thaw cycles.
Tissue homogenates - The preparation of tissue homogenates will vary depending upon tissue type. For this assay, tissues were rinsed in ice-cold PBS(0.01mol/L,pH 7.0-7.2) to remove excess blood thoroughly and weighed before homogenization. Minced the tissues to small pieces and homogenized them in 5-10 mL of PBS with a glass homogenizer on ice(Micro Tissue Grinders woks, too). The resulting suspension was sonicated with an ultrasonic cell disrupter or subjected to two freeze-thaw cycles to further break the cell membranes. After that, the homogenates were centrifugated for 5 minutes at 5000×g. Remove the supernate and assay immediately or aliquot and store at ≤-20oC.
Cell Lysates - Cells must be lysed before assaying according to the following directions.
1. Adherent cells should be detached with trypsin and then collected by centrifugation (suspension cells can be collected by centrifugation directly).
2. Wash cells three times in cold PBS.
3. Resuspend cells in PBS (1×) and the cells was subject to ultrasonication for 4 times (or Freeze cells at≤-20oC. Thaw cells with gentle mixing. Repeat the freeze/thaw cycle for 3 times.)
4. Centrifuge at 1500×g for 10 minutes at 2 - 8oC to remove cellular debris.
Sputum
Sputum must be treated before assaying according to the following directions. Mix the sputum with 0.1 % DTT (dithiothreitol) (two fold volume) and shake for 5 minutes at 37°C.
Add PBS (two-fold volume) to shake again for another 15-20 minutes. After Filtering through a 150 mesh wire net, remove particulates by centrifugation for 10 minutes at approximately 1500 rpm.
Feces
Try to collect dry stool, please avoid watery stool as it is difficult to prepare and it will influence the accuracy of detection. The weight is better to be over 50 mg, washing three times with PBS(final fecal mass: PBS Volume = 1: 9), Centrifuging 5000 x g for 10 minutes after ultrasonic pulverization (or crushed), get the supernatant for detection.
Saliva
Collecting sample in centrifuge tube, then freeze sample at -70°C for 1 hour. Thaw it on ice, and centrifuge at 2,000 × g for 10 minutes. Transfer clarified supernatant to clean tube for use in the assay.
Seminal
Collecting semen in sterile containers, normal sperm in vitro after injection was thick jelly, then it needs to liquefy by fibrinolytic enzymes at room temperature or 37 degree water bath. Then centrifuging at 4000 rpm for 10 min to seperate seminal plasma for detection.
Skin tissue
On the day of the assay, the biopsies were allows to thaw and 20-50 mg of wet ti ssue was homogenized with the aid of a Polytron homogenizer in ice-cold PBS contai ning a protease-inhibitor cocktail. The homogenates were centrifuged for 20 minutes at 10,000 x g to remove debris and insoluble material.
milk
The milk sample should be centrifuged at 12000g/min with 30min at 4°C, after remove the suspended lipid, and the deposit, then the samples could be used in detection or stored for later use.
Cerebrospinal Fluid, Bronchoalveolar Lavage Fluid
After collecting, centrifuge samples at 1000 × g for 20 min and get the supernatants.
The processing of sample is to collect target proteins, as proteins are generally easy to be denaturated, degraded, so the process should be as mild as possible. The storage of samples is also very important, especially be assured not to go freeze/ thaw cycles. And samples could be split charging. The storage time should be less than one week if at 4°C, should not exceed 1 month if at -20°C , -80°C should not exceed 2 months. Before assaying, bring samples to room temperature, and heating samples is forbidden. The processing of samples is the first step in a successful experiment. Above all, this a sketch on how to proceess common samples for the reference.
Urine - Aseptically collect the first urine of the day (mid-stream), voided directly into a sterile container. Centrifuge to remove particulate matter, assay immediately or aliquot and store at ≤-20 oC. Avoid repeated freeze-thaw cycles.
Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000×g at 2 - 8oC within 30 minutes of collection. Remove plasma and assay immediately or store samples in aliquot at -20oC or -80oC for later use. Avoid repeated freeze/thaw cycles.
Tissue homogenates - The preparation of tissue homogenates will vary depending upon tissue type. For this assay, tissues were rinsed in ice-cold PBS(0.01mol/L,pH 7.0-7.2) to remove excess blood thoroughly and weighed before homogenization. Minced the tissues to small pieces and homogenized them in 5-10 mL of PBS with a glass homogenizer on ice(Micro Tissue Grinders woks, too). The resulting suspension was sonicated with an ultrasonic cell disrupter or subjected to two freeze-thaw cycles to further break the cell membranes. After that, the homogenates were centrifugated for 5 minutes at 5000×g. Remove the supernate and assay immediately or aliquot and store at ≤-20oC.
Cell Lysates - Cells must be lysed before assaying according to the following directions.
1. Adherent cells should be detached with trypsin and then collected by centrifugation (suspension cells can be collected by centrifugation directly).
2. Wash cells three times in cold PBS.
3. Resuspend cells in PBS (1×) and the cells was subject to ultrasonication for 4 times (or Freeze cells at≤-20oC. Thaw cells with gentle mixing. Repeat the freeze/thaw cycle for 3 times.)
4. Centrifuge at 1500×g for 10 minutes at 2 - 8oC to remove cellular debris.
Sputum
Sputum must be treated before assaying according to the following directions. Mix the sputum with 0.1 % DTT (dithiothreitol) (two fold volume) and shake for 5 minutes at 37°C.
Add PBS (two-fold volume) to shake again for another 15-20 minutes. After Filtering through a 150 mesh wire net, remove particulates by centrifugation for 10 minutes at approximately 1500 rpm.
Feces
Try to collect dry stool, please avoid watery stool as it is difficult to prepare and it will influence the accuracy of detection. The weight is better to be over 50 mg, washing three times with PBS(final fecal mass: PBS Volume = 1: 9), Centrifuging 5000 x g for 10 minutes after ultrasonic pulverization (or crushed), get the supernatant for detection.
Saliva
Collecting sample in centrifuge tube, then freeze sample at -70°C for 1 hour. Thaw it on ice, and centrifuge at 2,000 × g for 10 minutes. Transfer clarified supernatant to clean tube for use in the assay.
Seminal
Collecting semen in sterile containers, normal sperm in vitro after injection was thick jelly, then it needs to liquefy by fibrinolytic enzymes at room temperature or 37 degree water bath. Then centrifuging at 4000 rpm for 10 min to seperate seminal plasma for detection.
Skin tissue
On the day of the assay, the biopsies were allows to thaw and 20-50 mg of wet ti ssue was homogenized with the aid of a Polytron homogenizer in ice-cold PBS contai ning a protease-inhibitor cocktail. The homogenates were centrifuged for 20 minutes at 10,000 x g to remove debris and insoluble material.
milk
The milk sample should be centrifuged at 12000g/min with 30min at 4°C, after remove the suspended lipid, and the deposit, then the samples could be used in detection or stored for later use.
Cerebrospinal Fluid, Bronchoalveolar Lavage Fluid
After collecting, centrifuge samples at 1000 × g for 20 min and get the supernatants.
The processing of sample is to collect target proteins, as proteins are generally easy to be denaturated, degraded, so the process should be as mild as possible. The storage of samples is also very important, especially be assured not to go freeze/ thaw cycles. And samples could be split charging. The storage time should be less than one week if at 4°C, should not exceed 1 month if at -20°C , -80°C should not exceed 2 months. Before assaying, bring samples to room temperature, and heating samples is forbidden. The processing of samples is the first step in a successful experiment. Above all, this a sketch on how to proceess common samples for the reference.
Urine - Aseptically collect the first urine of the day (mid-stream), voided directly into a sterile container. Centrifuge to remove particulate matter, assay immediately or aliquot and store at ≤-20 oC. Avoid repeated freeze-thaw cycles.
Cerebrospinal fluid (Csf)- Remove particulates by centrifugation and assay immediately or aliquot and store
samples at ≤-20oC. Avoid repeated freeze-thaw cycles.
Cell culture supernates and Other biological fluids - Centrifuge samples for 20 minutes at 1000×g. Remove particulates and assay immediately or store samples in aliquot at -20oC or -80oC. Avoid repeated freeze/thaw cycles.samples at ≤-20oC. Avoid repeated freeze-thaw cycles.
