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Autophagy (LC3)

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  1. How can I perform serum-starvation?
    We obtain serum-starved NRK cells as follows;
    1. Exchange the culture medium with Hanks' Balanced Salt Solution (serum-free).
    2. Incubate the NRK cells in the Hanks' Balanced Salt Solution(serum-free) for 2-4 hours.
    Starvation can also be induced using serum-free DMEM(Dulbecco's modified Eagle's medium), but amino acids in the DMEM reduce the level of starvation.
    Please note that optimal conditions may differ depend on the cell lines, so experimental conditions should be determined by end users.
  2. What percentage gel is suitable for detecting LC3 in Western Blotting?
    We recommend 15% gel. In 10% gel, bands of LC3-I and LC3-II overlap each other, making it difficult to distinguish these two bands.
  3. How can I detect LC3 band by Western Blotting?
    Please follow the protocol in the data sheet and check the points listed below.
    ● Make sure the sample buffer contains SDS. We recommend the use of SDS-PAGE sample buffer (Laemmli's sample buffer).
    ● When you detect LC3 with monoclonal antibodies, the washing step after blocking is necessary. Washing with 0.05% Tween-20/PBS (5 mins x 3 times) will produce stronger LC3-II signal.
    ● Cell suspension of Human LC3B transfected cells is available from MBL as a positive control in WB(Code No. PM036-PN).
  4. Do you have any information of how to interpret the LC3-I and LC3-II bands detected in Western Blotting?
    Please refer to the following research article. Interpretation of LC3 in Western Blotting is described in detail.
    Mizushima N, Yoshimori T. How to interpret LC3 immunoblotting. Autophagy. 3(6): 542-545, 2007 PMID:17611390
    Klionsky DJ, et al., Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes, Autophagy 4(2), 151 (2008) PMID: 18188003
    Klionsky DJ, et al., Guidelines for the use and interpretation of assays for monitoring autophagy, Autophagy 8(4), 445 (2012) PMID: 22966490
  5. What should I take notice when I perform immunocytochemistry using Anti-LC3 antibodies?
    We use Digitonin (Sigma, D141) in PBS for permeabilization and prepare freshly at a final concentration of 100 μg/mL. Triton X-100 is not suitable as a permeabilization solution for immunocytochemistry.
  6. Which fixatives are recommended for immunocytochemistry (IC)?
    We use 4% PFA/PBS. Methanol fixation or acetone fixation are not recommended.
  7. Which fixatives are recommended for immunohistochemistry (IH)?
    Please use 10% formalin solution (3.7% Formaldehyde) or 4% PFA/PBS.
  8. Can Anti-LC3 Antibodies by MBL be used for cryosections?
    The use on the cryosections has not been tested by MBL so far.
  9. Which antibody is recommended?
    Different antibodies are recommended depend on applications.
    Please check the information below.
    WB:Code No. M186-3, PM036
    IP:Code No. M152-3, PM036
    IC:Code No. M152-3, PM036
    FCM:Code No. M152-3, PM036
    IHC:Code No. PM036