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(無題) 削除/引用 No.6765-17 - 2018/04/02 (月) 02:22:16 - Tom Hi, mon-san, Do you remember the following paper? I am just curious the approach to purify such a minor protein. Thanks in advance, Tom > The concern I have is endogenous my protein X is really low in cells... You should use more cells if necessary. Suspension culture gives you a lot of cells easily. Former researchers purified transcription factors have use 100L culture or >1000 dished. Unbelievable!

(無題) 削除/引用 No.6765-16 - 2018/03/30 (金) 10:03:09 - mon Hi, Tom 1. Protein X might be strictly regulated quantitatively by non-ubiquitin system. 2. I hope your research will be fruitful. Good luck.

(無題) 削除/引用 No.6765-15 - 2018/03/30 (金) 09:47:12 - Tom Hi, mon-san, I just wanted to let you know my progress. 1. Protein X-V5-APEX2 was not detected by anti-V5 western blotting even in the treatment with 10 uM MG132 for 18 hrs. So I still have issue to express this protein in 293/293T cells. 2. Anyway, I went ahead to see if there is in vivo biotinylation. I successfully saw in vivo biotinylation (even the protein X was not detected tho)!! Thanks mon-san!

(無題) 削除/引用 No.6765-14 - 2018/03/23 (金) 05:49:02 - Tom Hi, mon-san, thank you so much for your advices. I just finished transfection and then tomorrow I will treat cells with MG132. > I wish a construct N-[signal peptide]-[APEX]-[X] -C will be functional... My protein of interest is type-I TM protein and definitely has signal peptide at the N-terminus. I did not think about your idea, but it seems worthy thinking. Thanks mon-san, Have a great weekend.

(無題) 削除/引用 No.6765-13 - 2018/03/22 (木) 16:49:07 - mon >So I do not know why this time protein X-V5-APEX2 was not detected...all fused tag was at C-terminus of protein X. Umm... Some Golgi- or ER protein have its localization signal at C-terminal. A big tag APEX might inhibits its function ??? I wish a construct N-[signal peptide]-[APEX]-[X] -C will be functional...

(無題) 削除/引用 No.6765-12 - 2018/03/22 (木) 09:48:04 - Tom Hi, mon, thanks for your comments! > >I could not detect protein X-V5-APEX2 by western blotting using anti-V5 mAb. > You said that X is in Golgi. Overexpressed proteins acted in ER and Golgi will often cause ER-stress to be ubiquitylated immediately and degraded. This time I overexpressed X-V5-APEX2 into 293T (but not in 293) by PEImax transfection. Again, anti-V5 did not give a band. Instead, anti-V5 gave a band from mito-V5-APEX2 transfected 293T cells. mito-V5-APEX2 was got from addgene as a positive control, indicating anti-V5 works and protein X-V5-APEX2 has issue to be expressed/maintained in cells. I also double checked DNA sequence of protein X-V5-APEX2 is right and no unexpected Stop codon there. Tomorrow I will test the effect of MG132 on the stability of protein X-V5-APEX2 as suggested. But, FYI, my protein X could be expressed in 293T cells if it is fused with myc-FLAG (pCMV6 from OriGene). Protein X-FLAG in pcDNA3.1 (+) was also expressed in 293T cells by same transfection way... So I do not know why this time protein X-V5-APEX2 was not detected...all fused tag was at C-terminus of protein X. I will think about changing plasmid backbone after finishing this expts. > 1.Wash excessive biotin phenyl. > Sorry, I have no knowledge.　I think excessive biotin phenyl will be removed by replacement of medium containing H202. Gotcha. > 2.Stimulate with H2O2 in serum free media. > I think usually it is not necessary when use of freshly prepared (diluted) H2O2-containing medium. But I used H2O2 form a flesh bottle (within 1 month) when stimulating cells. Again, gotcha. Thank you mon-san for great suggestion! I do appreciate your help. Tom

(無題) 削除/引用 No.6765-11 - 2018/03/18 (日) 11:26:23 - mon >I could not detect protein X-V5-APEX2 by western blotting using anti-V5 mAb. You said that X is in Golgi. Overexpressed proteins acted in ER and Golgi will often cause ER-stress to be ubiquitylated immediately and degraded. If MG132 treatment make X to be detect, ubiquitylated X is degraded by proteasome. If so, you should use weak~mild promoter instead of strong CMV promoter. 1.Wash excessive biotin phenyl. Sorry, I have no knowledge.　I think excessive biotin phenyl will be removed by replacement of medium containing H202. 2.Stimulate with H2O2 in serum free media. I think usually it is not necessary when use of freshly prepared (diluted) H2O2-containing medium. But I used H2O2 form a flesh bottle (within 1 month) when stimulating cells.

(無題) 削除/引用 No.6765-10 - 2018/03/18 (日) 03:14:32 - Tom I have a few concerns about labeling with biotin. 1. Do I need to wash excessive biotin phenyl from dishes before adding H2O2? 2. Long time ago when I was engaged in H2O2-induced apoptosis experiments, I generally stimulate cell line with H2O2 in serum free media otherwise H2O2 did not induce apoptosis in my hand. So I am wondering if after incorporation of biotin phenyl I may need to wash the dish before adding H2O2 so that it could certainly emerge phenyl radical enough. What do you think, mon?

(無題) 削除/引用 No.6765-9 - 2018/03/18 (日) 03:08:27 - Tom Hi, mon (continued) Before using 30% H2O2 original bottle, I put a drop of original 30% H2O2 solution on my arm and it certainly injured my arm with itches, indicating H2O2 is functional. Biotin phenyl was purchased from APExBIO (#A8011) and I dissolved into DMSO. So it should be fine... DNA sequence of Protein X-V5-APEX2 fusion protein plasmid was confirmed. Although I could not detect protein X-V5-APEX2 by western blotting using anti-V5 mAb. I just plated 293T cells (but not 293 cells used yesterday), and then I will schedule to transfect again protein X-V5-APEX2 for 48 hrs (but not 24 hrs yesterday) to see if biotinization certainly occurs... But based on Nature protocol, 24 hrs after transfection is the time to go. Otherwise, over-expression causes aggregation of expressed proteins said Nature protocol. I hope it works...the data will be obtained by Monday, will update. Thanks again mon for your great suggestions. It was certainly helpful.

(無題) 削除/引用 No.6765-8 - 2018/03/18 (日) 03:05:30 - Tom Hi, mon, Again, I do appreciate your great suggestions! OMG, really? It's really incredible! It's actually practical suggestions. I do keep in mind once I start such expts. >You can also use a combination method of DSP-crosslinking/immuno-precipitaion after APEX2-labeling. Yeah, that what I am thinking about:) Thanks again mon-san, you are awesome scientist! Indeed, yesterday I just started V5-APEX2-fused protein X experiments. I transiently expressed Protein X-V5-APEX2 into 293 cells. After 24 hrs, I added phenyl biotin (500 uM) for 30 min. And then, I additionally added final 1 mM H2O2 for 1 min. After that, I washed the cells with PBS containing NaN3, Na Ascorbate and Trolox as quenchers. Then, I lysed the cells with 1% triton X-100 based lysis buffer followed by centrifuge and adding SDS sample buffer. Today, I tried to detect biotinized proteins in lysates with HRP-streptavidin. But I could not see any bands. By contrast, I could detect biotinized ladder in positive control lane (biotinized isotype IgG). That means HRP-avidin works fine. The problem was biotinization did not occur.

(無題) 削除/引用 No.6765-7 - 2018/03/17 (土) 08:19:31 - mon > The concern I have is endogenous my protein X is really low in cells... You should use more cells if necessary. Suspension culture gives you a lot of cells easily. Former researchers purified transcription factors have use 100L culture or >1000 dished. Unbelievable! Your could obtain the more specific DSP-linked proteins purified with streptavidin-beads after concentration with your specific antibody. And you should concern that Lys/Arg (trypsin-cleavable sites) will modified by closslinkers if use MS/MS to identify the target proteins. > BTW, do you think isolation of membrane fraction in my APEX2 approach may still have chance to get golgi membrane proteins biotinized? Because even if cytosolic proteins are labeled much, it could be excluded by isolating of membrane fraction. I think possible but unexpected difficulty will be. You can also use a combination method of DSP-crosslinking/immuno-precipitaion after APEX2-labeling. Good luck.

(無題) 削除/引用 No.6765-6 - 2018/03/17 (土) 06:11:58 - Tom Hi, mon, I do appreciate your great suggestion! I was impressed with alternative approach. HRP has not been used to label intracellular proteins because its reducing environment of cytosol, but approach you mentioned is perfect as I don't need to care about redox state, thus HRP is functional! Awesome. Wow, you often do that? That really helps. I definitely think about this. The concern I have is endogenous my protein X is really low in cells... BTW, do you think isolation of membrane fraction in my APEX2 approach may still have chance to get golgi membrane proteins biotinized? Because even if cytosolic proteins are labeled much, it could be excluded by isolating of membrane fraction. What do you think? Thanks, Tom

(無題) 削除/引用 No.6765-5 - 2018/03/17 (土) 05:20:06 - mon Hi Tom, Your aim may be carried out with an alternative "in vitro" biotin-labling method as follows, 1. Crosslink intracellular proteins with DSP 2. Permeabilize cells 3. Proximity-dependent biotin labelling by HRP-labled antibody (or 1st specific Ab & HRP-labled 2nd Ab) with biotin tyramine. 4. Harvest biotinylated proteins with DTT which cleaves DSP linker. I often use the procedure #1~4 (w/o #3) as an alternative Immuno-precipitation method for detecting weakly binding proteins.

(無題) 削除/引用 No.6765-4 - 2018/03/17 (土) 04:38:11 - mon If activity of the engineered APEX2 is too strong, you may use the original APEX (I think a codon-optimized version is suitable).

(無題) 削除/引用 No.6765-3 - 2018/03/17 (土) 04:29:08 - Tom Hi, mon! I am pleased to see reply! OMG! Thanks for attached! I see, it seems tons of cytosolic proteins are labelled by cytosolically exposing APEX2... I guess this tech may not be able to identify Golgi membrane proteins specifically which I wanted to do ...

(無題) 削除/引用 No.6765-2 - 2018/03/17 (土) 04:02:53 - mon Hi, Tom Intracellular biotin-labling by AEPX2 could be possible. See a reference: sciencedirect.com/science/article/pii/S2211124716305083

in vivo labeling/BirA and APEX system 削除/引用 No.6765-1 - 2018/03/13 (火) 09:25:51 - Tom Hi, there, I am just wondering if someone gives me suggestions on in vivo labeling. I want to identify interacting proteins with my protein of interest X. The X is type-1 membrane protein localized at Golgi membrane. Recently a couple of paper reports in vivo labeling using BirA or APEX2, and I am also interested in that technologies. My concern is that if I fuse APEX2 on the C-terminus of X, APEX2 is exposed to cytosol. Does that mean cytosolic proteins are also potentially labeled with biotin? In other word, is it really feasible approach to identify interacting proteins? Or it just identifies proximal but not interacting proteins? Appreciate your great helps in advance, Tom

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