Total protein extraction from adipose tissue and cells

Summary

Extraction of total protein from adipose tissue and cells

Principle

Currently, water-oil emulsions in biological samples are the most difficult to separate. Centrifugal tube columns with unique surface properties with pore sizes and optimized surfactant-free buffer systems can quickly and effectively separate water-oil emulsions from adipose tissue homogenates. The extraction buffer has a lower freezing point than the oil in the adipose tissue, and the adipose tissue homogenate passes through the centrifuge column quickly separating the aqueous phase from the oil phase, with no loss of total protein in the tissue. The protein yield can reach 2-3 mg/ml, which is much higher than other methods.

Appliance

Various fields of life sciences

Operation method

Column Method Minute TM Total Protein from Adipose Tissue and Cells

Materials and Instruments

【Material】Fresh or frozen adipose tissue;
[Reagents] Reagent kit; protein extraction powder; buffer A, B, C;

Move

1. Place Buffer A, centrifuge tube column and receiver tube cannulae on ice to pre-cool.2. weigh 50-80 mg of fresh or frozen adipose tissue, place it on several layers of paper towels, and remove some of the oil from the tissue by squeezing with your thumb and forefinger. Using tweezers, place the tissue into the 1.5 ml centrifuge tube provided in the kit and weigh 100 mg of protein extraction powder to add to the sample. Add 50ul of Buffer A. 3.3. Twist and grind the tissue sample with a grinder bar for 1-2 minutes to form a paste. Add 200-300ul of Buffer A and continue to grind the sample for 30 seconds. If the starting tissue amount is small (20-40 mg) add 100-150ul of Buffer A. 4. Cover with a lid.4. Cover and centrifuge at 2000 rpm for 1 minute. Transfer the supernatant to a centrifuge column placed in a receiver tube (the grinding rod can be reused, cleaned with an alcohol wipe and air dried).5. Incubate at -20°C with the lid open for 15-20 minutes. (Make sure that the refrigerator temperature is near -20°C, otherwise refer to the FAQ below.)6. Immediately after incubation, centrifuge at 2000 rpm with the lid open for 1-2 minutes. Dispose of the centrifuge column to obtain total protein from adipose tissue. The extracted protein contains a small amount of insoluble material that is not a water-soluble cellular fraction. It can be diluted and used directly in an ELISA to detect water-soluble proteins. Solubilized insoluble proteins can also be resuspended in Buffer B or C for downstream applications:A. Add 1/10 Buffer B to the extracted protein solution to make a denatured protein solution (for SDS-PAGE, Western and other applications) orB. Add 1/10 Buffer C to the extracted protein solution to make a non-denatured protein solution (for IP, ELISA and other applications) orC. Dissolve in 2X 2D Gel Sample Buffer for 2D analysis.

Caveat

NOTE: Buffer A contains components that may be infectious for mass spectrometry analysis. If extracted proteins are to be used for mass spectrometry, dialyze the buffer before extraction or precipitate the proteins by TCA.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/