Endoplasmic Reticulum Enrichment Extraction Kit

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Endoplasmic Reticulum Enrichment Extraction Kit [NBP2-29482] - This kit is routinely tested and standardized using 0.5 gram of mouse liver tissue.
Endoplasmic Reticulum Enrichment Extraction Kit [NBP2-29482] - ATF6 (3 ug/mL) and GAPDH (0.25 ug/mL). ATF6 is an ER stress-regulated transmembrane transcription factor. The blots were probed with ATF6, stripped and ...read more
Endoplasmic Reticulum Enrichment Extraction Kit [NBP2-29482] - ATF6 (3 ug/mL) and GAPDH (0.25 ug/mL). ATF6 is an ER stress-regulated transmembrane transcription factor. The blots were probed with ATF6, stripped and ...read more
Endoplasmic Reticulum Enrichment Extraction Kit [NBP2-29482] - Ontology analyses of differentially expressed proteins in mouse cohorts.

Product Details

Summary
Kit Type
Extraction Kit

Order Details

Endoplasmic Reticulum Enrichment Extraction Kit Summary

Description
Isolating the ER may seem difficult due to its size and complex association with other components of the cytosol. The Endoplasmic Reticulum Enrichment Kit is a key tool that provides the necessary reagents together with an optimized protocol required to gently isolate and enrich for ER from tissues.
Kit Type
Extraction Kit

Applications/Dilutions

Application Notes
The kit is routinely tested using 0.5 gram of freshly isolated mouse liver tissue as the starting material. We typically get the following yields:
Total ER fraction: 350 uL (12 mg/mL = 4.2 mg) or
Rough ER fraction: 300 uL (9 mg/mL = 2.7 mg)
These yields are based on using the 0.5 gram starting material for either isolating the Total ER fraction or the Rough ER fraction. If the starting material is divided at Step C and both the Total and Rough ER fractions are isolated, then yields will be approximately 1/2. Please note that all yields are approximate and may vary depending on the tissue type used and other variables. Use in Western blot reported in scientific literature (PMID 23904601).
Publications
Read Publications using
NBP2-29482 in the following applications:

Packaging, Storage & Formulations

Storage
Storage of components varies. See protocol for specific instructions.

Kit Components

Components
  1. 5X Isosmotic Homogenization Buffer (30 mL)
  2. 1X Suspension Buffer (30 mL)
  3. 10X PBS (2 x 50 mL)
  4. 5X Calcium Chloride Solution (60 mL)
  5. 100X Protease Inhibitor Cocktail (2 x 0.75 mL)

Notes

Additional Reagents/Equipment required but not provided:
Sterile water
Standard balance
High-speed centrifuge (Beckman GS-15R centrifuge
Beckman Avanti J30I centrifuge with JS24 rotor or equivalent)
Compatible centrifuge tubes
Glass homogenizer (15-mL Wheaton tube and Pestle or equivalent for 1 gram of tissue sample)
Microcentrifuge tubes

Alternate Names for Endoplasmic Reticulum Enrichment Extraction Kit

  • ER Enrichment

Background

The Endoplasmic Reticulum (ER) is a membranous labyrinth that extends throughout the cytoplasm of the cell as a single continuous network of flattened sacs and tubules. The ER accounts for more than half the total membrane in eukaryotic cells and 10% of a cell's total volume, thus representing one of the largest organelles. It plays a critical role not only in lipid and protein biosynthesis but is central to protein modification, folding, and assembly. The ER is generally characterized as being devoid of membrane-bound ribosomes, (smooth ER), or studded with ribosomes (rough ER); each differing in structure, appearance, and function.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Kits are guaranteed for 6 months from date of receipt.
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Publications for Endoplasmic Reticulum Enrichment Kit (NBP2-29482)(20)

We have publications tested in 5 confirmed species: Human, Mouse, Rat, Mouse/Rat, Plant.

We have publications tested in 2 applications: WB, in vitro.


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WB
(8)
in vitro
(1)
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Human
(1)
Mouse
(3)
Rat
(1)
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Showing Publications 1 - 10 of 20. Show All 20 Publications.
Publications using NBP2-29482 Applications Species
Chan LC, Li CW, Xia W et al. IL-6/JAK1 pathway drives PD-L1 Y112 phosphorylation to promote cancer immune evasion J. Clin. Invest. Jul 15 2019 [PMID: 31305264]
Sanvee GM, Bouitbir J, Krahenbuhl S Insulin prevents and reverts simvastatin-induced toxicity in C2C12 skeletal muscle cells Sci Rep May 15 2019 [PMID: 31092879] (WB, Mouse) WB Mouse
Cha JH, Yang WH, Xia W et al. Metformin Promotes Antitumor Immunity via Endoplasmic-Reticulum-Associated Degradation of PD-L1 Mol. Cell Aug 16 2018 [PMID: 30118680] (Human) Human
Komatsu S, Hashiguchi A. Subcellular Proteomics: Application to Elucidation of Flooding-Response Mechanisms in Soybean. Proteomes. Feb 27 2018 [PMID: 29495455] (Plant) Plant
Aumailley L, Roux-Dalvai F, Kelly I et al. Vitamin C alters the amount of specific endoplasmic reticulum associated proteins involved in lipid metabolism in the liver of mice synthesizing a nonfunctional Werner syndrome (Wrn) mutant protein. PLoS ONE. Mar 1 2018 [PMID: 29494634] (Mouse)

Details:
The Novus ER enrichment kit was used to collect endoplasmic reticulum fractions from mouse livers
Mouse
Park SH, Kang MK, Choi YJ et al. alpha-Asarone blocks 7B-hydroxycholesterol-exposed macrophage injury through blocking elF2alpha phosphorylation and prompting beclin-1-dependent autophagy. Oncotarget. Jan 31 2017 [PMID: 28088783] (Mouse) Mouse
Wang X, Komatsu S. Gel-Free/Label-Free Proteomic Analysis of Endoplasmic Reticulum Proteins in Soybean Root Tips under Flooding and Drought Stresses. J. Proteome Res. Jun 2 2016 [PMID: 27224218]
Shin D, Feltri ML, Wrabetz L. Altered Trafficking and Processing of GALC Mutants Correlates with Globoid Cell Leukodystrophy Severity. J. Neurosci. 2016 Feb 10 [PMID: 26865610]
Wang X, Komatsu S. Plant subcellular proteomics: Application for exploring optimal cell function in soybean. J Proteomics. 2016 Jan 22 [PMID: 26808589]

Details:
ER enrichment kit was used for isolation of Endoplasmic Reticulum from root tips of soybean (see full text of this publication for detailed & modified protocol).
Son SH, Seko A, Daikoku S et al. Endoplasmic reticulum (ER)-targeted, galectin-mediated retrograde transport using a HaloTag carrier protein. Chembiochem. 2016 Jan 15 [PMID: 26773807]

Details:
ER enrichment kit was used for the isolation of Endoplasmic Reticulum from PC 12 (rat adrenal pheochromocytoma) D cells which were pre-incubated with recombinant protein carrying X (G1-ps-Halo(X)-KDEL; 6.0 uM.
Show All 20 Publications.

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Product General Protocols

View specific protocols for Endoplasmic Reticulum Enrichment Kit (NBP2-29482): Find general support by application which include: protocols, troubleshooting, illustrated assays, videos and webinars.

Video Protocols

WB Video Protocol

FAQs for Endoplasmic Reticulum Enrichment Kit (NBP2-29482). (Showing 1 - 8 of 8 FAQs).

  1. Regarding the Endoplasmic Reticulum Enrichment Kit, what is the common amount of ER when using this kit for cultured cells?
    • We have evaluated this Kit in tissues. We have not yet tested this Kit in cell lines. However, this kit has been described for use with cell lines in citation #2 in the product manual.
  2. To lysis cultured cells, should the tight or loose version of the glass homogenizer from Wheaton grinder be used? Also, does 90000 x g in the protocol correlate to max or average RCF with the Beckman centrifuge speed specification? With the JS-24.38, what RPM is recommended?
    • We use the Wheaton grinder loose version. With our centrifuge we set the speed at 90000 g, our rotor doesn't have options to specify max or average settings, there is just a setting. But if the options on your rotor are for max or average, then I would imagine average would be the one to choose, but either one might work just as well.
  3. What is the number of cells required for using this kit? If a sample includes detergent, will that effect the kit? Which fraction includes cell membrane fraction and Golgi body fraction after centrifugation several times?
    • We have used the kit with tissue. We haven't used the kit with cells. The customer will need to empirically determine the amount of cells needed. We unfortunately do not have any information on whether or not detergents will affect the usefulness of this kit. This kit is designed for endoplasmic reticulum enrichment. It is not designed for preservation of the Golgi and cell membrane fractions. The kit has been developed based on information in the general references listed in the manual provided with the kit.
  4. Could you tell us if this kit can be used to enrich from culture cells (e.g. Caco2 or HEK293)? If yes, please let us know detailed protocol and recovery ratio about that.
  5. Does NBP2-29482 contain any polyclonal antibodies?
    • Our kit NBP2-29482 does not contain any polyclonal antibodies. The components list for this kit can be found on page five of the following manual: http://resources.novusbio.com/manual/Manual-NBP2-29482-2449737.pdf
  6. I used the ER enrichment kit, however the total ER fraction I collected at the procedure of C2 contained much of lysosome. (I confirmed it on WB of Lamp2 and enzyme assay of beta-galactosidase.) I would like to be informed if you have  troubleshooting for this condition. The centrifuge I used is Beckman XE90, the rotor is SW41Ti.The cell applied is 4x10^6 cells of COS7.
    • With regard to your inquiry, my lab thinks that if the lysosomes are the same density and size as the ER population, they will be present in the ER collection. They recommend additional homogenization/lysis time on your cell population at the beginning of your enrichment process. They also think that further dilution could help as well. If possible, also try to add another centrifuge step to help remove other organelles prior to the high speed spin. We think these suggestions should help to eliminate the presence of the lysosomes in your ER collection.
  7. Could you tell us if the kit components contain ascorbic acid? #KC-415 1X Suspension Buffer, #KC-121 100X Protease Inhibitor Cocktail (PIC)
    • The kit components for our kit NBP2-29482 do not contain any ascorbic acid. The only component that contains any acids is KC-121, and this component contains 1% acetic acid.
  8. I used Endoplasmic Reticulum Enrichment Extraction Kit (Cat. No. NBP2-29482) to extract the rough endoplasmic reticulum (RER) of plant mulberry leaves. After extraction, according to the kit procedure, I did a Western blot. The results showed that the GRP78 band in the supernatant after the final step of centrifugation was brighter than in the precipitate. What is the principle of adding calcium chloride when the kit is used to rough the endoplasmic reticulum? Because I want to try to increase the calcium chloride concentration to increase the yield of rough endoplasmic reticulum, What is the concentration of calcium chloride in the kit? 
    • We recommend you to review paper entitled "A rapid calcium precipitation method of recovering large amounts of highly pure hepatocyte rough endoplasmic reticulum" by Hamilton et al ( published in Journal of Lipid Research Volume 40, 1999) wherein the modified calcium precipitation method had been described in more detail. The 5x CaCl2 solution is 40mM. You may be able to improve your results by using ice-cold 1x (8mM) CaCl2 and adding it drop-wise to the solution. The GRP78 protein is a ER marker but it will mark both smooth and rough ER, so the fact that there is plenty of GRP78 in your solution does not have to be an indication that the extraction was not complete. There are just smooth ER vesicles in the solution, which won't get precipitated until you spin it down at min 90 000xg.

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