Blue Native PAGE (BN-PAGE) behind the scenes

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  • Опубликовано: 24 дек 2024

Комментарии • 34

  • @purplepanda9794
    @purplepanda9794 3 года назад +1

    Thank you! Absolute life-saver

  • @anushab4347
    @anushab4347 2 года назад +3

    Hi Brianna! I just recently stumbled upon your channel and i wanna tell ya its helping me BIGGG time!! Nothing is more powerful than sharing one's valuable knowledge and experience with others. After watching some of your protein separation videos, it begs me the question why is normal native page method used? If the intention is to separate proteins based on size and charge, you have 2D PAGE utilizing isoelectric focussing followed by SDS PAGE. If however the intention is to only obtain proteins in its native conformation, then you have blue native page as you don't have to worry whether your sample contains positively charged proteins or not (which i think is the main drawback of normal native page technique). So what is the usecase of normal native page according to you or is it redundant? correct me if i'm wrong. Lots of love, thank you!

    • @thebumblingbiochemist
      @thebumblingbiochemist  2 года назад +1

      Thanks so much! I'm so happy you've found my content helpful. I really do feel my education and experience is an immense privilege and it would be selfish of me not to try to help share it! For PAGE, your proteins somehow need to be negatively-charged (not positively-charged). With SDS-PAGE, the SDS adds the negative charge (in addition to denaturing the protein), and with blue-native PAGE, the CBB does the job. With normal native PAGE, your protein would have to have some negative charge, but cytosolic proteins typically are slightly acidic, so you don't need to worry about it. The problems only arise when proteins are basic, which can be an issue especially with membrane proteins, and proteins that reside in the nucleus or mitochondria. Here's an article you might find interesting in this regard: bmcmolcellbiol.biomedcentral.com/articles/10.1186/s12860-019-0221-4. So blue native is often used there. But if you don't need to go through the extra hassle of having the dye and stuff, there's no reason to add it. Especially because it has the potential to interfere with interactions. Hope that helped answer your question!

  • @naturelover-ip5ng
    @naturelover-ip5ng Год назад

    Hi Brianna. Thank you for demonstrating how to run a blue native PAGE. I have one question. Biorad electrophoresis manual has a recipe of native sample buffer that contains bromophenol blue. It will be easier to pinpoint your samples with the blue when loading them into the gel. Is there a specific reason that you don't use that sample buffer?

    • @thebumblingbiochemist
      @thebumblingbiochemist  11 месяцев назад

      The sample buffer always has dye in it

    • @naturelover-ip5ng
      @naturelover-ip5ng 11 месяцев назад

      @@thebumblingbiochemist but you just added glycerol to your samples in your video. Did I miss something?

    • @thebumblingbiochemist
      @thebumblingbiochemist  11 месяцев назад

      Oh sorry - it's been a while since I did this and I got mixed up. Dye will definitely make things easier! The key thing though is just to make sure not to use normal sample buffer, which has the denaturant.

  • @ynr21
    @ynr21 8 месяцев назад

    Hi, thanks for the great video. Can you please share a link or name of the protein marker that you used ? Thanks.

    • @thebumblingbiochemist
      @thebumblingbiochemist  8 месяцев назад +1

      I think it was a NativeMark ladder: www.thermofisher.com/order/catalog/product/LC0725

    • @ynr21
      @ynr21 8 месяцев назад

      @@thebumblingbiochemist thank you so much 😊

  • @nordoliverturro8252
    @nordoliverturro8252 8 месяцев назад

    thank you for your work it is amazing. We "PhD student in IBMB Barcelona" on Friday afternoon made an improvised native blue. including comassie in the loading buffer (0.25% (w/v)) and in the gel (0.002% (w/v)) but not in the runing buffer in the cathode part as you do. The samples (3 different proteins that are supposed to form complex) ran differently in the wells alone than in the ones with the supposed complex, so the result does not seem bad. A colleague told me that I was not doing it right and he showed me your video ,so I want to repeat the experiment in your style. In the end I think that your method is much less aggressive for the protein since you do not expose it to high concentrations of commasi and you also do not put commassie in the polymerization of the gel (I don't know how that will affect it).

  • @cosmicpc2886
    @cosmicpc2886 Год назад

    Can someone explain why this procedure is important. I purely want to understand why separating proteins is important.

    • @thebumblingbiochemist
      @thebumblingbiochemist  Год назад +1

      It allows you get an idea about what's in a solution. More here: bit.ly/gel_vs_blot

  • @andrewoliveira3645
    @andrewoliveira3645 2 года назад

    Awesome video! How did you prepare 100x dye? Thanks in advance

    • @thebumblingbiochemist
      @thebumblingbiochemist  2 года назад +2

      Thanks so much! My lab mate actually made it a while ago and said he got the recipe online but needs to look back at his notes. I will let you know if he tells me more. Sorry

  • @nasabghazal8345
    @nasabghazal8345 2 года назад

    Thanks for your video!! I’ve tried the native mark from invitrogen multiple times and I can’t see it in no way shape or form. How did you get it to show up on your gel??!! Thanks so much

  • @dancingbiologist2688
    @dancingbiologist2688 3 года назад

    thank you for this very informative video!😊

  • @dino4605
    @dino4605 2 года назад

    Many thanks for your useful video! I have a same question with Andrew. How did you prepare 100x dye? And could you please tell me what kind of native marker product you used?

    • @thebumblingbiochemist
      @thebumblingbiochemist  2 года назад +2

      Hi. I never got that dye recipe from my colleague sorry! And I believe it was a NativeMark ladder

    • @dino4605
      @dino4605 2 года назад

      @@thebumblingbiochemist Thanks a lot for your kind reply 😁

  • @vidyajayanti7459
    @vidyajayanti7459 2 года назад

    Hey, cool video. I am having issues with destaining the blue dye. I feel like its preventing my antibodies from binding because I don’t get any bands on my developer. What do you do to deatain and for how long? Ive used methanol 100% and 8% acetic acid but the membrane is still very blue. Also when i am running my page using the anode and cathode buffer all biorad powerpacs show an error when i run at 150v. Ive tried a couple of them with different electrodes as well. And last question, ive bought the invitrogen ladder for native page, how to stain it, do you cut it from your membrane?? Any help will be muchhh appreciated

    • @thebumblingbiochemist
      @thebumblingbiochemist  2 года назад

      Hi - I've only run it once and that was a while ago, so I'm sorry but I don't have answers! But I only included the dye in the beginning (~20 min) to avoid overstaining. Good luck!

    • @micronerd
      @micronerd 5 месяцев назад

      ​@@thebumblingbiochemist This response might be a life-saver, also saw someone discussing removing the stain at about 1/3rd gel length on ResearchGate. Faced exactly such over-staining issue today while attempting to extablish a BN-PAGE procedure at my lab, so definitely will try again with removal of the stained cathode buffer 😅

  • @alcyonecrucis
    @alcyonecrucis Год назад

    love it !

  • @rommathedex_657
    @rommathedex_657 2 года назад

    Subbed!