Endoplasmic Reticulum Isolation and ER Membrane Protein Purification

Creative Proteomics offers endoplasmic reticulum and endoplasmic reticulum membrane protein extraction and purification services for animal tissues, cells and yeast and help you obtain purified endoplasmic reticulum and proteins for subsequent assays.

Endoplasmic reticulum (ER) is a network of membranous sacs that form interconnected tubules, vesicles and cisternae. The ER membrane is a continuation of the outer nuclear membrane, and plays a critical role in cellular functions, including protein synthesis, production of steroids, insertion of membrane proteins, transport of proteins, glycoproteins, and lipoproteins, and so forth. There are two types, rough ER and smooth ER. The rough ER is close to nucleus, and contains ribosomes, where proteins synthesis. And the smooth ER is composed of tubules and vesicles, and is the center where lipid and membrane protein synthesis. What's more, there are various disease caused by malfunction of the ER stress, including diabetes, inflammation, and neurodegenerative disorders, and so on. To have better understanding numerous processes of cell activities, such as protein transport, protein synthesis, lipid metabolism, etc., it is the necessary to isolate rough and smooth ER.

Advantages of Our Endoplasmic Reticulum and ER Membrane Protein Isolation and Purification Services:

• Rich experience in ER separation and purification.
• Obtained pure ER with low impurities and intact membrane.
• ER can be isolated and purified from tissues, cultured cells, yeast and plant cells.
• Protein can be extracted from ER without contamination.
• We will conduct purity verification tests to ensure that the isolated ER and ER membrane protein reaches the target purity, otherwise there is no charge.

We Provide the Following Services, including but not Limited to:

• Single organelle separation and purification
• Crude ER (microsomes), rough ER and smooth ER separation and purification
• ER membrane protein extraction
• Ribosome separation from rough ER

Methods of Endoplasmic Reticulum Isolation and ER Membrane Protein Purification:

1. Single organelle - ER isolation and purification
Rough ER is a complicated network of tubular and cisternal structures that are converted into small vesicles when cells are homogenized. It is impossible to obtain morphologically intact rough ER. The rough ER has a unique buoyant density, which is strictly affected by the number of associated ribosome per unit of membranous area. To get fractions of ER, including rough ER and smooth ER, Density Gradient Centrifugation is conducted. According to whether they are associated with ribosomes or not, ER vesicles will migrate to different densities. Through serial centrifugations, nuclei, cell debris, mitochondria, and lipids to obtain a post mitochondrial fraction can be removed. In order to further separate rough ER or smooth ER from the ER fractions, Discontinuous Sucrose Density Gradients are used. Since Mg²⁺ is one main determinant of association of ribosomes with the rough ER, the Density Shift approach can be adopted by removal of Mg²⁺ from the medium, which results in a reduction in the ER membrane density and a shift by density gradient centrifugation. The specialized and optimized protocol will be designed based on your experimental goals and subsequence purposes.

The purity requirements for ER fractions purity determine the appropriate method required. Depending on your sample types and subsequent experiment, different detergents and methods will be optimized to meet your needs.

2. Isolation and purification of multiple organelles
If you need multiple organelle proteins, then we will use the organelle fractionation method to separate and enrich the organelle proteins you need.

3. Extraction of ER membrane proteins
We use an optimized method that combines Differential and Percoll Gradient Centrifuges to extract ER membrane proteins. The extracted ER membrane proteins can be used for subsequent immunoblotting, proteomics, and so forth.

Delivery

Experimental protocol, purity analysis report, extracted ER and purified protein sample.

Want to learn more about ER membrane protein characterization, protein-protein interaction, protein synthesis and transport analysis and other analysis services? We provide one-stop analysis of endoplasmic reticulum proteomics.

References

1. Sabatini D.D. Subcellular Fractionation of Rough Microsomes. Cold Spring Harb Protoc; doi:10.1101/pdb.top074567.
2. Sabatini D.D. Preparation of Crude Rough Microsomes from Tissue Culture Cells. Cold Spring Harb Protoc; doi:10.1101/pdb.prot079996.
3. Williamson C.D., Wong D.S., Bozidis P., et al. Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane and Detergent Resistant Membrane Fractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts. Curr Protoc Cell Biol. 68: 3.27.1–3.27.33.
4. Dallner, G., Isolation of rough and smooth microsomes – general. Methods in Enzymol. 31,191-201 (1974).

* For Research Use Only. Not for use in diagnostic procedures.