Minute™ Detergent-Free Protein Extraction Kit for Microbes with Thick Cell Walls

Protein extraction from cell wall-containing microbes is a frequent procedure in bio-research Labs. The methods for protein extraction from these microbes are usually harsh, tedious and not sufficiently reliable. YD-016 provides a detergent-free, rapid and gentle way for extracting proteins from microbes with thick and strong cell walls. These microbes include but not limited to yeast, filamentous fungus, gram positive and negative bacteria, insect eggs and microalgae. This kit contains optimized detergent-free and EDTA-free protein extraction buffers. The whole procedure takes less than 10 min to complete and the protein yield is in the range of 2-4 mg/ml with un-biased protein representation. This kit features a single tube protocol and works well for both log phase and stationary phase microbes. The materials provided are sufficient for 50 extractions.

Application
Proteins extracted with this kit can be used for many downstream applications such as SDS-PAGE analysis, Western blotting, IP. ELISA and enzyme activity assays and proteomic analysis. The buffers are compatible with IMAC resins for his-tagged protein purification

 

Kit components
1. 20 ml buffer A
2. 20 ml buffer B
3. 5 g protein extraction powder
4. 4 pestles for 1.5 ml microcentrifuge tube
5. 1.5 ml microfugr tube X 50

SDS-PAGE (12%) profiles of extracted proteins by detergent-free extraction buffers.
Lane 1: S. cerevisiae
Lane 2: L. lactis (Gram+)

Important Product Information
The use of protease inhibitors is not necessary prior to extraction. However if downstream application takes significant amounts of time or the protein extract will be stored for longer period of time, addition of protease inhibitors to extraction buffer is recommended. For determination of protein concentration, BCA kit is recommended. To study protein phosphorylation, phosphatase inhibitors shoμld be added to lysis buffer prior to use.

Protocol

1. Harvest microbes of interest by centrifugation in a 1.5 ml microfuge tube. Make sure that
the wet volume of pellet is between 20-30 μl. The volume can be easily estimated by comparing it to a 1.5 ml tube with 30 μl water.
2. Wash the pellet with one ml water by centrifuging at top speed in a microcentrfuge for 2 min. Remove supernatant completely. Resuspend the pellet in 50 μl buffer A. Weigh out 80-90 mg protein extraction powder and add to the bottom of the tube (try not to touch the wall of the tube. This can be done by weighing out the powder in a piece of folded wax paper and pour the powder to the bottom of the tube).
3. Grinding the tube repeatedly with the pestle provided for about 2 min with twisting force. Add 50 μl buffer A and 100 μl buffer B to the same tube and continue to grind for about thirty seconds (note: The pestle is reusable, for cleaning simply soak it in bleach, rinse with water and dry it with paper towel). Cap the tube and vortex vigorously for 10 seconds.
4. Centrifuge the tube at top speed for 3-4 min. Transfer the supernatant to a fresh tube (this is detergent-free total protein). If more protein is desired repeat step 3-4 one more time. Typically protein yield is 2-3 mg/ml. Extracted protein can be stored at -80oC for future use.

Application tips: The final protein yield is proportional.

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