ProteinExt TM Mammalian Total Protein Extraction Kit

   ProteinExtTM Mammalian Total Protein Extraction Kit
ProteinExtTM Mammalian Total Protein Extraction Kit provides a simple and fast way to extract total protein (including cytoplasmic, membrane and nuclear protein) from mammalian cells and tissues. This kit does not require ultracentrifuge. The extracted protein is suitable for SDS-PAGE, Western Blot and ELISA.  

Cat. No.	DE101
Specification	DE101-01	100 ml
Storage:	at -20 o C for one year
Notes	Prior to use, add PMSF into TPEB.
	We suggest aliquoting TPEB for storage after initial thawing.
	All steps should be carried out on ice or at 4 o C.
	For total protein extraction from tissues, 1-2 ml glass homogenizer is supplied by user.
	If protein quantification is required, we suggest BCA method with TransGen Easy II Protein Quantitative Kit (BCA), Cat. No DQ111.

  Kit Contents

Component	DE101-01
Total Protein Extraction Buffer (TPEB)	100 ml

  Procedure
a. Cultured Cells
1. Harvest 0.5-2×10 7 cells and discard the culture media; wash the cells with 1 ml of pre-chilled PBS by twice and centrifuge at
500×g for 5 minutes, gently discard the supernatant.
2. Add 1 ml of TPEB to cell pellet (please adjust the volume based on the ratio at 0.5-2×10 7 cells/ ml TPEB). Mix thoroughly by
vortexing, and incubate on ice for 30 minutes with vortexing at every 10 minutes.
3. Centrifuge at 14,000×g, 4 o C for 10 minutes.
4. Gently collect the supernatant (total protein) and store at -80 o C or directly proceed to downstream application.
b. Tissues
1. Cut 20-100 mg of tissues into small pieces, wash minced tissues with 1 ml of pre-chilled PBS by twice and centrifuge at
500×g for 5 minutes, gently discard the supernatant.
2. Add 1 ml of TPEB to tissues and mix thoroughly by vortexing. Transfer the suspension to a pre-chilled glass homogenizer and
homogenize the tissue by 6-10 strokes.
3. Gently transfer the suspension to a new 1.5 ml microcentrifuge tube, incubate on ice for 30 minutes with vortexing at every
10 minutes.
4. Centrifuge at 14,000×g, 4 o C for 10 minutes.
5. Gently collect the supernatant (total protein) and store at -80 o C or directly proceed to downstream application.