Blue Native PAGE (BN-PAGE) behind the scenes
Summary
TLDRThis video script delves into the intricate process of Blue Native PAGE, a technique used to separate protein complexes without denaturing them, preserving their natural structures and interactions. The narrator shares their hands-on experience, from preparing the gel to running the electrophoresis, highlighting the unique aspects of Blue Native PAGE, such as using Coomassie Blue dye to impart a negative charge on proteins for migration. Despite the non-informative results, the process's aesthetic appeal and the learning experience are emphasized, showcasing the experimental journey's value beyond mere outcomes. The script also touches on subsequent steps like staining and potential analyses, offering insights into the broader context of protein study techniques.
Takeaways
- 🔬 Blue Native PAGE (Polyacrylamide Gel Electrophoresis) is a technique used to separate protein complexes without denaturing them, allowing for the observation of multimer formations.
- 🧬 Unlike SDS PAGE, Blue Native PAGE does not unfold proteins but instead uses Coomassie Blue dye to impart a negative charge, enabling their movement through the gel based on size.
- 🔵 The Coomassie Blue dye gently binds to proteins and complexes without breaking them up, facilitating their migration through the gel.
- 🎨 The process results in visually striking blue gels, adding an aesthetic aspect to the scientific experiment.
- ⚡ The technique involves using electricity to move proteins through the gel, similar to SDS PAGE, but maintains protein complexes intact.
- 📊 Pre-cast gels with varying concentrations (e.g., 4-20%) and specialized ladders are utilized to accommodate the unique properties of proteins in Blue Native PAGE.
- 💧 Additional steps include careful sample preparation with glycerol to prevent samples from floating out of wells and ensuring a tight seal in the gel apparatus to avoid leaks.
- 🕒 After initial running, the inner buffer containing the dye is replaced with a fresh, undyed buffer to prevent over-dyeing and potential issues.
- 🔍 Post-run, proteins can be visualized directly if concentrated enough, or through staining techniques like Coomassie or silver staining for more sensitivity.
- 🧪 The experiment serves both an educational and exploratory purpose, demonstrating the application of Blue Native PAGE and providing hands-on experience with the technique.
Q & A
What is the primary purpose of using native PAGE over SDS PAGE?
-The primary purpose of using native PAGE over SDS PAGE is to separate protein complexes without unfolding the proteins, thereby preserving their native structure and allowing for the observation of multimers and protein complexes.
How does SDS PAGE differ from native PAGE in terms of protein treatment?
-SDS PAGE unfolds proteins and applies a uniform negative charge across them, facilitating their separation by size. Native PAGE, on the other hand, does not unfold proteins, allowing them to maintain their natural structures and complexes.
What is the role of the comassie blue dye in blue native PAGE?
-In blue native PAGE, comassie blue dye gently binds to proteins and their complexes without disrupting them, imparting a negative charge that enables their migration through the gel.
Why might a protein not migrate through a gel in SDS PAGE?
-A protein might not migrate through a gel in SDS PAGE if it does not have a negative charge under the conditions used, such as if the protein is inherently basic and positively charged, it won't be pulled through the gel without additional treatment.
What is the significance of replacing the dye with a fresh, undyed buffer during a blue native PAGE run?
-Replacing the dye with fresh, undyed buffer during a blue native PAGE run prevents over-dyeing and potential issues related to excessive staining, ensuring clearer results and better visualization of the proteins.
How does the presence of glycerol in the sample affect its migration in blue native PAGE?
-Glycerol is added to the samples to increase their density, ensuring they sink into the wells and do not float out, which is crucial for maintaining sample integrity during the electrophoresis process.
What is the purpose of using a special ladder in blue native PAGE?
-A special ladder is used in blue native PAGE because the normal ladders for SDS PAGE are denatured and would not behave correctly under native PAGE conditions. The special ladder is designed to migrate properly without denaturing.
What does the presenter mean by 'lazy staining' the gel, and why was it chosen?
-Lazy staining refers to a quick and simple staining method used by the presenter, chosen for convenience despite less informative results. It involves staining the gel directly, without prior destaining, to quickly visualize proteins.
What are the implications of spilling dye into the outer chamber of the gel apparatus?
-Spilling dye into the outer chamber can lead to unintended dye migration and contamination, affecting the clarity of the visualization and potentially the interpretation of the results, as it might obscure the visibility of the wells and migration lanes.
What are some post-run analyses that can be performed on proteins separated by blue native PAGE?
-Post-run analyses include staining the gel for visualization, conducting a second dimension PAGE for further separation, performing western blotting for specific protein identification, or analyzing the proteins via mass spectrometry for a comprehensive identification of the protein components.
Outlines
🔬 Introduction to Blue Native PAGE
This segment introduces Blue Native PAGE, a method used to separate protein complexes without unfolding them, preserving their natural structures and interactions. Unlike SDS PAGE, which denatures proteins and imparts a uniform negative charge, Blue Native PAGE relies on the gentle binding of Coomassie Blue dye to proteins. This binding does not disrupt the protein complexes but grants them a negative charge, enabling their migration through a gel under an electric field. The speaker highlights the visual appeal of the process, expressing fascination with the blue-colored gel and the anticipation of seeing their protein of interest. Despite uninformative results, the speaker values the learning experience, emphasizing the importance of experimentation.
🧪 Conducting Blue Native PAGE: Process and Observations
This paragraph details the step-by-step process of conducting Blue Native PAGE, including sample preparation, gel loading, and running the experiment. The speaker uses a precast gel and a special loading buffer to prevent proteins from floating out of the wells. The experiment begins without the dye, followed by loading samples and adding glycerol to stabilize them. After initial setup, the gel is run at 100 volts, with the speaker adjusting conditions to prevent over-dying and to ensure a clean separation of protein bands. The technique's visual appeal is noted, with the transition from dye to an undyed buffer highlighted as a critical step. Despite challenges, such as accidental dye spillage, the experiment proceeds, showcasing the unique aspects of Blue Native PAGE, including the preservation of protein complexes and the aesthetic aspect of the blue gel.
📊 Results Interpretation and Further Analysis
The final segment focuses on the aftermath of the Blue Native PAGE process, discussing ways to interpret and further analyze the results. The speaker mentions staining techniques for visualizing proteins within the gel, including quick stain and silver stain for more sensitive detection. They address the potential of the gel to reveal protein bands directly due to the Coomassie dye's presence. The speaker also outlines post-experiment options like cutting out specific bands for further analysis, conducting a second dimension PAGE for complex disassembly, or transferring proteins for western blot analysis. For identifying unknown proteins, mass spectrometry offers an unbiased approach. This segment underscores the versatility of Blue Native PAGE in protein analysis and the breadth of post-experiment techniques available for detailed protein characterization.
Mindmap
Keywords
💡Native PAGE
💡SDS-PAGE
💡Blue Native PAGE
💡Coomassie Blue dye
💡Electrophoresis
💡Protein complexes
💡Multimers
💡Glycerol
💡Voltage
💡Staining
Highlights
Introduction to native PAGE, a technique to separate protein complexes without unfolding them, maintaining their structural integrity.
Comparison between SDS PAGE and native PAGE, highlighting the latter's ability to analyze protein complexes and multimers.
Explanation of how SDS PAGE unfolds proteins and imparts a negative charge, a process not involved in native PAGE.
Introduction to blue native PAGE, a method that uses Coomassie Blue dye to impart a negative charge on proteins without unfolding them.
Demonstration of the experimental process, including preparation and considerations for running a blue native PAGE.
Explanation of the use of a special ladder for blue native PAGE, different from the one used in SDS PAGE.
Description of sample preparation and loading for blue native PAGE, highlighting the importance of glycerol in sample wells.
Technical tips on ensuring a tight seal in the gel apparatus to prevent leakage during the electrophoresis process.
The initial steps in running the gel, including setting voltage and the significance of not over-dying the gel.
Explanation of how to prevent over-dying by replacing the dye in the buffer partway through the process.
Aesthetic appreciation of blue native PAGE, emphasizing its unique visual appeal in the laboratory.
Adjustments made during the electrophoresis run, including voltage changes and the decision-making process involved.
Discussion on the post-run staining process, including options for quick staining or more sensitive staining methods like silver staining.
Insight into the various applications of gel results, including second dimension PAGE and protein identification techniques.
The unbiased approach to protein identification through mass spectrometry, enabling discovery without prior knowledge of the proteins present.
Transcripts
just looks so cool
like
somewhere in there is my protein
yesterday i told you about native page
in which we separate protein complexes
together um through a gel so it's like
sds page and that you're using a gel to
separate
proteins by their size using electricity
um to get them to move through the gel
but unlike sds page you're not unfolding
the proteins
and so this keeps complexes together so
you can see if proteins are like forming
multimers and that sort of thing
but
the scs it doesn't just like unfold the
proteins it also gives them a negative
charge and so if your protein doesn't
have a negative charge um
at least at the ph that's used in the
the buffer then your protein is not
going to have that urge to go it's not
going to have that pull to go through
the gel and so it won't
so if you have one of these like basic
proteins which is positively charged
what you can do is you can use this
technique called blue native page in
which you use um comassie blue dye
um and this dye is going to like gently
bind to the proteins and the complexes
so it's not going to break them up but
it will give them a negative charge and
this is going to let them travel through
the gel
um and so yesterday i told you about the
theory and today i want to show you how
i actually did it because it's really
it's blue and it's kind of cool looking
um and really that's
well
the science is cool too but it it was
really pretty and i had never seen
something like that um and so i thought
i'd share oh my results weren't uh very
informative but i'm still glad i did it
because it's one of those things that
i've always wanted to know how to do and
now i do you never know until you try
i'm using like a precast four to twenty
percent gel
my one extra slicing buffer
i'm going to do this
so at this point this is just without
the
no dye yet
now i am going to
load my samples
so you have to use like a special ladder
the normal ladders are like for sbs page
um so they're like denatured and stuff
okay so now i'm going to load my samples
so you can see they're clear because
they don't have the sds um die
you have 20 final glycerol so that they
won't like float out of the wells
thankfully these pre-runs have them
numbered because you can't see very well
without the dye
i'm just going to fill the empty lanes
with like a 20
um so that
it doesn't run wonky
because i only have eight samples and so
if you have like uneven you have like a
bunch of empty wells that's gonna
and so i'm gonna add like two mils
[Music]
and two the center chamber which is
where the cathode is the inner chamber
of the cathode chamber so i'm gonna put
it into the bottom of the
yeah so you want to make sure that you
have a good tight seal
when you are
preparing the inner buffer inner gasket
make sure that you have it like tightly
in there or else it's gonna leak out so
you should see it's just in the center
and now i'm just going to gently
i apparently need to charge my
pipette names a little
and now
i can start
the run
so
i'm gonna start it
like 100 volts
and you can't it's hard to like see if
anything's happening because you can't
really see the bubbles rising when
there's a bunch of blue
but it looks like it's running and
now i am going to um let it run for like
20 minutes or so until the dye and stuff
like the proteins everything enters the
gel
um and then i'm going to stop it remove
the inner buffer so remove the blue
stuff and put in just like fresh uh
non-dyes having stuff and that's gonna
prevent it from getting like over dyed
and uh
causing problems and stuff
and right now i'm going to get out of
the cold room
i actually spilt some dye into the outer
chamber
um
that's why it's blue it's not it
shouldn't look blue like if you don't
want it to things be leaping and you
don't need the blue on the outer because
you can see the eyes were in there so my
protein should be in there
and now i'll turn it back on
and hopefully
it'll work
so blue page looks kind of freaking
awesome
so the reason why it's kind of like you
have that band is because you
start it with uh the dye
and then you replace it with like fresh
um
undyed buffer
so you don't like over dye it and stuff
um
and so i'm running
um and my protein somewhere in there
hopefully i will be able to see it
increase the voltage now
time to turn it off yeah so after you
can do a bunch of different things um so
i just stained mine to look at it
um and i lazy stained it um so basically
when because you have like the dye in
the gel
even if though you replace the buffer
part way through you're still gonna have
like a dark dye band um and so
thankfully like
that's below the proteins i was
interested in um so i didn't really
worry about de-staining it before i just
stuck into some quick stain
um if you have like a ton of protein in
there you might be able to see some
bands without even staining because when
you're staining it you're adding komasi
um but so if you wanted to like really
sensitive stain you can even like silver
stain but for those things you're going
to want to like you stain the gel first
um
and so there's like
these stain solutions and stuff like
acetic acid and stuff and then you can
restain it um and that's also going to
like when you stain it that way like you
destain and stuff you're like fixing the
gel the proteins in place so that they
like stay um
like they precipitate in place and so
they're not going to like diffuse out of
the gel
but if you are going to do something
further like you're going to take the
bands out and
like
do something or you're going to do a
second dimension page or whatever
there's different things that you're
going to do and so you're not going to
like want to fix them in place um but
basically look at the protocols if you
want to know more about the various
things you could do like a second
dimension page which i talked about
yesterday where you kind of like cut
that band out you soak into some sds and
denaturing reagent to denature the
proteins but they're stuck in place in
the gel
and then you kind of take that band and
then you like turn it on the side and
run it through sds page and so you're
separating the components of the complex
um they'll be in the same column
and you can also do things like a
western blot to transfer it out of the
gel and onto a membrane and then prove
it for specific um proteins but for that
you need to know what proteins you're
looking for you can also like just send
like take the bands and give them to a
mass spectrometer person
and max spectrometrist um and ask them
to identify the proteins filled like um
take them the sample and then they can
like identify the peptides in the
protein um based on like the sequence um
so that's like an unbiased approach so
you don't have to know what you're
looking for and it'll say oh this is
what it is
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