Hello Guys,
Im doing a compartmental protein extraction for cytoplasm, nuclear and cell wall proteins . In the protocol I have to use beads based homogenizer. Do these homogenizer destroys cell walls ? I was thinking to use sanitation as our lab have no bead based homogenizer. Can you please suggest any other method
Thanks,
Amended on 9 September 2018
I like to use simply ultra-sonic oscillation method (please see files; SEC column 300 A silica and HepG2 fucoidan). Cooling on ice bath is important. Imput the sonic power by pulse may be better to prevent the elevation of temperature. The time for oscillation should be within 5 min which is sufficient to make membrane structure (please see file; Ala transport). I have used 3 min of sonication for preparing cell or tissue homogenates (please see file; HPLC-Surf-SEC protein determination method).
I have studied using autopsies and biopsies of hepatocellular carcinoma (HCC). It is essential to cut the tissue as far as we can by knife or scalpel with putting on threefold gloves. Cancer tissue is dangerous since virus and microbes are richly present. HCC is caused by enveloped +ssRNA virus of HIV-1 (human specific retrovirus) and HCV (human specific Flavivirus) (please see file again; HepG2 fucoidan).
Human breast cancer seems to be caused by enveloped Flavivirus of Dengue virus/DENV or Zika virus/ZIKV or enveloped Togavirus of Sindbis virus/SINV or Chikungunya virus/CHIKV plus enveloped retrovirus of HIV-1 (our unpublished estimation).
Human cervical cancer seemes to be by enveloped human retrovirus of HIV-1 and enveloped +ssRNA virus of Murine coronavirus/MCoV, Canine coronavirus/CCoV, or Avian infectious bronchitis virus/IBV/Avian coronavirus/ACoV. Therefore, Cervical cancer seems not to be caused by non-enveloped dsDNA virus of Human papillomavirus/HPV at all.
By the way, biopsies of liver diseases has many invaded microbes (our unpublished observation).
Liver (Pseudo liver cancer) has virus, Rickettsia conorii, and Bacteroides nodosus at 18.9% of protein among tissue proteins.
LC tissue (with leprosy) has virus, Agrobacterium tumefaciens, and Mycobacterium leprae at 15.6% of protein among tissue proteins.
HCC tissue (with PBC) has virus, Mycobacterium bovis, and Schizosaccharomyces pombe at 30.2% of protein among tissue proteins.
LC tissue (named as No.6) has virus and Salmonella enterica at 23.9% of protein among tissue proteins. HCC tissue (named as No.6) has virus only at 17.7% of protein among tissue proteins.
Therefore, I have wrapped the tube (please use Eppendorf's plastic tube (glass tube frequently has been broken), and heat-resistant food-wrap) during sonication. Please wear a mask and put on a pair of glasses.
Which cell type you are using? Bacterial cells are having relatively rigid cell wall but beat-beater will cause cell wall rupturing. If you do not have beads beater or homogenizer, you mix the cells pellet (after centrifugation) with zirconia beads or glass powder and grind manually in a pastle motar. Separate cellular organalles by differential centrifugation thereafter.
Thank you for your answer shamsher. Im working with breast cancer tumor tissues. Can i use sonicator ?
Surely you can use sonicator but I am afraid while working with a cancer cell line tiny droplets shall form during use of sonic waves and you might need a biosafe system or use sonicator inside a biosafe hood!
Avoid exposure to aerosol during sonication of transformed cells and bear a face mask!
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