UPX Universal Protein Extraction Kit FAQ

Basics

What Does the UPX Universal Protein Extraction Kit Do?

Protein Discovery’s UPX Universal Protein Extraction Kit is designed to overcome the soluble-protein bias of traditional extraction methods, to extract proteins at high efficiency, and to solubilize the entire proteome as completely as possible. It is compatible with downstream proteomics applications such as 1-D PAGE analysis and size-based fractionation using the Gelfree 8100 Fractionation System.

What Kinds of Cells and Tissues Does the UPX Universal Protein Extraction Kit Work On?

Protein Discovery’s UPX Universal Protein Extraction Kit provides unbiased protein extraction and work with high efficiency on mammalian organ tissues such as heart, liver, brain, and kidney, and mammalian-derived cultured cell lines, and bacteria. Tissues can be frozen or freshly harvested.

How Can I Tell How Much Protein Has Been Extracted?

The UPX Universal Protein Extraction Buffer contains SDS and reducing agents. For accurate determination of total protein concentration, use a protein assay compatible with detergents and reducing agents.

Can I Analyze Protein Activity?

The UPX Universal Protein Extraction Kit was designed for research in which high-yield unbiased extraction, complete solubilization, and maximum depth-of-proteome coverage are high priorities. Proteins extracted using the UPX Kit are reduced and denatured in the process. In some instances, it may be possible to treat the extracted proteins in a manner that would result in restoration of functional activity, but such a workflow is not supported by Protein Discovery.

Special Applications

Can I Use IEF to Separate Proteins Extracted using the UPX Kit?

The lysis buffer included with the UPX Universal Protein Extraction Kit contains SDS. Therefore proteins extracted using the UPX Kit cannot be separated by IEF (isoelectric focusing).

Can UPX buffer be used to resolubilize immunoprecipitated proteins?

If the targeted protein has already been isolated from the antibody, then UPX buffer will work well as a resolubilizing agent. However, UPX buffer for this application could be problematic if introduced at the wrong time. UPX contains both a strong solubilizing detergent and a reducing agent, which increase extracted protein yields dramatically. If UPX buffer were to be used to extract protein from bead-bound antibody, the disulfide bonds of the antibody would be reduced and the final sample would be contaminated by light/heavy chain antibody.

Protein Discovery recommends the following procedure for resolubilization of immunoprecipitated proteins that have not been isolated from the antibody: Make a solution of 2% SDS in Tris (50 mM, pH 7.5). Use a minimal volume of this solution to elute your targeted protein from the bead-bound antibody. Add an equal volume of UPX buffer to the eluate (1:1 v/v) and follow the UPX Kit protocol to carry out the reduction with heating. If preparing the sample for fractionation, load 10-25 μg protein on the Gelfree 8100 Fractionation System.

Purchasing Considerations

How Many UPX Universal Protein Extraction Kits Do I Need?

The UPX Universal Protein Extraction Kit comes with 50 mL UPX Universal Protein Extraction Buffer. The UPX Extraction Kit protocol specifies 10 mL UPX buffer per gram of tissue or 0.3 g of wet cell pellet. Order as many UPX Universal Protein Extraction Kits as you need to complete the protein extraction experiments you plan to perform within the shelf life of the kit.

What is the Shelf Life?

The shelf life of UPX buffer is six months when stored at 4 °C.

What Else Do I Need to Carry Out the Protocol?

In addition to a UPX Universal Protein Extraction Kit, you’ll need:

  • Protease Inhibitor Cocktail
  • Dounce Homogenizer
  • Centrifuge Tubes
  • Vortex/Shaker
  • Centrifuge
  • Water Bath/Hot Plate set to 100 °C.