Minute Plasma Membrane Protein Isolation Kit
Contents
1. Minute Plasma Membrane Protein Isolation Kit User Manual v2 Invent Biotechnologies Inc Minute Plasma Membrane Protein Isolation Kit Catalog number SM 005 Description Invent Biotechnologies Minute plasma membrane protein isolation kit is composed of optimized buffers and protein extraction filter cartridges with 2 0 ml collection tubes The kit is designed to rapidly isolate native total membrane proteins organelle membrane proteins and native plasma membrane proteins from cultured mammalian cells or tissues This kit can sequentially separate cellular components into four fractions nuclei cytosol organelles and plasma membrane Due to the use of protein extraction filter cartridges the membrane protein isolation is simple easy and user friendly with high yield Unlike many commercial membrane preparation kits that require large amount of starting cells 5 millions and up This kit offers wide range of starting cells 1 50 millions sample The buffers are detergent and EDTA free A Dounce homogenizer or a tissue blender is not needed The procedure can be completed in less than 45 min Applications The kit is designed to rapidly isolate native membrane proteins from cultured cells or tissues for applications such as SDS PAGE immunoblottings ELISA IP membrane protein structure analysis 2 D gels enzyme activity assays and other applications This kit provides the most rapid method currently available for preparation2. of native membrane proteins Buffer Formulations Proprietary Kit components 50 preps 25 ml buffer A 10 ml buffer B 50 protein extraction filter cartridges 50 collection tubes with cap 4 plastic rods Tissue dissociation beads DAARWN gt Storage Store Buffer A and Buffer B at 20 C upon arrival Additional Materials Required 1X PBS Vortexer Table Top Microcentrifuge Invent Biotechnologies Inc 8961 Aztec Dr Eden Prairie MN 55347 Tel 866 817 7611 Fax 952 400 6889 www inventbiotech com 1 Minute Plasma Membrane Protein Isolation Kit User Manual v2 Invent Biotechnologies Inc Important Information 1 Read the entire procedures carefully Thaw buffer A and buffer B completely invert the bottles a few times and place them on ice Chill protein extraction filter cartridge with collection tube on ice prior to use All centrifugation steps should be performed at 4 C in a cold room or in a refrigerated mirocentrifuge To study protein phosphorylation phosphatase inhibitors such as PhosStop from Roche should be added to buffer A prior to use The use of protease inhibitor cocktails is optional It is recommended to use BCA Protein Assay Kit for determination of protein concentration Pierce Cat 23227 Membrane Protein Isolation Procedures A 1 2 3a 3b 4 Isolation of Total Membrane Proteins Place the filter cartridges in collection tubs and incubate on ice For cultu
3. on if further isolation of plasma membrane proteins is desired Isolation of Plasma Membrane Proteins Resuspend the total membrane protein fraction from step 6 in 200 ul buffer B by repeatedly pipetting up and down or vortexing Centrifuge at 10 000 rpm for 5 min at 4 C The pellet contains organelle membrane proteins Carefully transfer the supernatant to a fresh 2 0 ml microcentrifuge tube and add 1 6 ml cold PBS Mix by inverting the tube a few times Centrifuge at 16 000 rpm for 15 30 min longer centrifugation will improve yield Discard the supernatant and save the pellet isolated plasma membrane proteins Typically 10 300 ug plasma membrane proteins can be obtained Pellet of plasma membrane proteins can be dissolved in 20 200 ul detergent containing buffers of your choice such as 0 5 Triton X 100 in PBS Troubleshooting Problem Solution Low protein yield Increase starting cell numbers Increase incubation time to 10 min step3 Low protein activity Keep lysate cold add protease inhibitors Retention of cell lysate in protein filter cartridge Reduce amount of starting material or increase after 30 seconds of centrifugation centrifugation time to 2 min Invent Biotechnologies Inc 8961 Aztec Dr Eden Prairie MN 55347 Tel 866 817 7611 Fax 952 400 6889 www inventbiotech com 3
4. red cells collect 1 50 X 10 cells by low speed centrifugation 500 600 X g 5 min Go to step 3a For tissue samples go to step 3b Note For isolation of plasma membrane proteins from cultured cells see below it s recommended to use 20 50 X 10 cells Wash cells once with cold PBS Remove supernatant completely and resuspend the pellet in buffer A 200 ul for a starting cell number less than 5 million and 500 ul for a starting cell number greater than 5 million Incubate the cell suspension on ice for 5 10 min Vortex the tube vigorously for 10 30 seconds Immediately transfer the cell suspension to the filter cartridge Go to step 4 For tissue samples place a piece of fresh tissue 10 30 mg or frozen tissue 20 30 mg in a filter cartridge Add 200 ul buffer A to the filter and grind the tissue with a plastic rod for one min by pushing the tissue against the surface of the filter repeatedly with twisting force Note if you are working with skeletal or cardiac muscles it is recommended to add 100 120 mg tissue dissociation beads to the filter prior to grinding Add 300 ul buffer A to the same filter cartridge mix by pipette up and down a few times and incubate the tube on ice with cap open for 5 min Go to step 4 Note The presence of a small amount of un homogenized tissue will not affect the quality of the sample The plastic rod is reusable For cleaning wipe it with 75 alcohol or rinse it with distilled water Cap the fil
5. ter cartridge and centrifuge at 14 000 rpm for 30 seconds Invent Biotechnologies Inc 8961 Aztec Dr Eden Prairie MN 55347 Tel 866 817 7611 Fax 952 400 6889 www inventbiotech com 2 Minute Plasma Membrane Protein Isolation Kit User Manual v2 Invent Biotechnologies Inc Optional For cultured cells it is recommended to resuspend the pellet in collection tube from step 4 transfer the cell suspension to the same filter and spin at 14 000 rpm for 30 seconds Re passing the cells through the filter can increase the yield by 20 30 Discard the filter and resuspend the pellet by vigorously vortexing for 10 seconds Following procedures separate total cellular components into four fractions nuclei cytosol organelles and plasma membrane Centrifuge at 3000 rpm for one min the pellet contains intact nuclei Transfer the supernatant to a fresh 1 5 ml microcentrifuge tube and centrifuged at 4 C for 10 30 min at 16 000 rpm longer centrifugation time will increase yield Remove the supernatant this is the cytosol fraction and save the pellet this is the total membrane protein fraction including organelles and plasma membranes Store the pellet at 70 C or dissolve it in detergent containing buffers of your choice The yield is typically 10 500 ug sample You may stop here if isolation of plasma membrane proteins is not needed Continue to step 7 for plasma membrane protein isolation Don t freeze total membrane protein fracti
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