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Product Details
| Cat # +Size | K268-50 |
|---|---|
| Size | 50 assays |
| Species Reactivity | Mammalian |
| Applications | Western blotting, 2-D gels, and enzyme analyses, etc. The entire procedure takes less than 1 hour. |
| Features & Benefits | • Simple procedure • Fast and convenient • The Membrance Protein Extraction Kit provides an easy and complete protocol for isolating integral membrance proteins efficiently from cultured mammalian cells. |
| Kit Components | • Homogenize Buffer • Upper Phase Solution • Lower Phase Solution • Protease Inhibitor Cocktail |
| Storage Conditions | -20°C |
| Shipping Conditions | Gel Pack |
| USAGE | For Research Use Only! Not For Use in Humans. |
Details
The Membrane Protein Extraction Kit provides optimized buffers and reagents for effective extraction of membrane proteins from mammalian tissues and cells. Unlike other available procedures that can only extract the total cellular membrane proteins (combinations of plasma and organelle membrane proteins), BioVision’s kit was designed to not only extract the total cellular membrane proteins, but also purify the plasma membrane proteins specifically. The procedure offers consistent yield and high purity (over 90%). Membrane proteins prepared using the kit can be utilized in a variety of applications, such as Western blotting, 2-D gels, and enzyme analyses, etc. The entire procedure takes less than 1 hour.
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Can the proteins extracted with the Membrane Protein Extraction Kit be used for mass spectrometry analysis?
Proteins extracted can be used for a variety of downstream applications including mass spectrometry. However, we have not specifically tested the proteins in mass spectrometry applications.
What is the expected yield with this kit?
The expected membrane protein recovery is 100 ug/50 million cells. The expected amount of cytosolic protein is 3-30 mg depending on cell types.
My yield is very low. Please advise.
A few suggestions regarding your questions: A few ways to get efficient yield:1. Use ~10(E8) cells to start.2. Make sure most of the cells are lysed.3. Keep on ice for overnight at step C 13 after 5 volume dilution.4. After overnight incubation, transfer to new tubes just before the centrifugation, so that you can get a tight visible pellet.
Does the kit work on the protein complex of plasma membrane protein?
In the kit procedure, getting total cellular membrane protein procedure should not dissociate protein complex. You can use the kit to get total cellular membrane proteins, then do IP. If you further purify the plasma membrane, the yield will be low.
Does the kit works after we co-immunoprecipitate the protein?
The protocol we have with this kit gives native proteins. It may depend on how strong the association of the proteins. The phase solution we use with the kit contains PEG and Dextran. We do not use any denaturing detergents.
In the final plasma membrane pellet, are the membranes still intact (i.e. are there lipids present)?
Most unbound lipids are removed during the process.
What type of membrane protein does this kit extracts?
The kit mainly extracts trans-membrane proteins. The efficiency should depend on the property of individual protein. It should extract multiple trans-membrane domain proteins well.
Do you know if your membrane protein isolation kit can be used to isolate membrane proteins together with membrane associated proteins.
This will depend on the association strength. If the association with the trans membrane protein is strong, it can be isolated.
Is it possible to homogenize the cells with a needle and syringe instead of a Dounce homogenizer for this protocol?
Using a Dounce homogenizer is actually the most efficient way of lysing the cells. You can try using sonication along with the needle and syringe, but ensure efficient lysis before you proceed to the next step.
Can we use frozen samples with this assay?
Fresh samples are always preferred over frozen samples. However, frozen samples can also be used, provided, they were frozen right after isolation, were not freeze thawed multiple time (for which we recommend aliquoting the samples before freezing) and have been frozen for relatively short periods.
What is the exact volume of sample required for this assay?
There is no specific volume we can recommend for the amount any sample to be used since it is completely sample concentration and quality based. You have to do a pilot expt with multiple sample volumes to determine the optimal volume which gives a reading within the linear range of the standard curve. Please refer to the citations for this product to see what other clients have used with similar sample types.
Do you have trial sizes of this kit?
Unfortunately, we do not have trial sizes of this kit available. However, if you are based in the US or Canada, we will give you a 10% off list price introductory discount on its purchase price. If you are based out of this area please contact your regional BioVision distributor.
What is the shelf life of this kit?
This kit is good for 12 months from the date of shipment in the unopened form when stored at the appropriate temperature and appropriate conditions. After opening and reconstitution, some of the components in this kit are good for 2 months at -20°C. Please refer to the datasheet for storage information and shelf life of each of the components.
Why are my standard curve values lower than those shown on the datasheet?
There are multiple factors which influence the signals like the incubation times, room temperature, handling etc. In general, to increase the value of the standards, you can increase the incubation time. As long as the standard curve is linear, it should be fine to use, since all of your samples will also be measured under the same conditions on this curve.
How do I normalize my samples against protein concentration
You can use a protein quantitation assay on the supernatants you get from cell/tissue lysates or with any other liquid sample in the assay buffer.
Can we purchase individual components of this kit?
Yes, you can purchase any of the kit's components without the whole kit. Please refer to the component Cat #s mentioned on the datasheet for ordering.
Can we use an alternate buffer for sample preparation (cell lysis, sample dilutions etc)?
Our assay buffers are optimized for the reactions they are designed for. They not only contain some detergents for efficient lysis of your cells/tissue, but also contain some proprietary components required for the further reactions. Therefore, we highly recommend using the buffers provided in the kit for the best results.
Should I dissolve the final pellet in a buffer imperatively containing 0.5% Triton X-100 or PBS alone would be enough? What is the exact role of Triton?
In general, detergents help to solubilize and stabilize membrane proteins preventing aggregation.
Is the plasma membrane protein fraction compatible with downstream ELISA assays?
We haven't tested it. You might need to do the standardization on your end.
Ai-Qin Wang, Mechanisms by which fibroblast growth factor 20 improves motor performance in a mouse model of Parkinson’s disease. Neural Regen Res., Aug 2019; 30964070.
Jingjing Wang, Clathrin-mediated Endocytosis of Alpha-1 Antitrypsin is Essential for its Protective Function in Islet Cell Survival. Theranostics, May 2019; 31281523.
Soochi Kim, Activation of LXRɑ/β by cholesterol in malignant ascites promotes chemoresistance in ovarian cancer. BMC Cancer, Dec 2018; 30526541.
Sun, Yan-Yan et al. (2016) Surface expression of hippocampal NMDA GluN2B receptors regulated by fear conditioning determines its contribution to memory consolidation in adult rats Sci Rep. 2016 Aug 4;6:30743. doi: 10.1038/srep30743.
Sin et al., Acute Treatment of Resveratrol Alleviates Doxorubicin-Induced Myotoxicity in Aged Skeletal Muscle Through SIRT1-Dependent Mechanisms. J Gerontol A Biol Sci Med Sci, Oct 2015; 10.1093/gerona/glv175.
For more citations of this product click here
