Blue Native PAGE (BN-PAGE) behind the scenes

Summary

This 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

00:00

🔬 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.

05:03

🧪 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.

10:06

📊 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.

Keywords

💡Native PAGE

Native PAGE (Polyacrylamide Gel Electrophoresis) is a technique used in biochemistry to separate proteins based on their native, or non-denatured, state and molecular weight without breaking down their complex formations. In contrast to SDS-PAGE, which denatures proteins to separate them, Native PAGE maintains the proteins' natural shapes and complexes, allowing the observation of protein-protein interactions, such as the formation of multimers. This method is essential for studying proteins in their functional forms, as mentioned in the script when discussing the separation of protein complexes.

💡SDS-PAGE

SDS-PAGE stands for Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis. It's a common laboratory technique used to separate proteins based on their molecular weight. SDS is a detergent that denatures proteins, meaning it unfolds them into linear chains and coats them with a negative charge, ensuring they move towards the positive electrode during electrophoresis. The script contrasts SDS-PAGE with Native PAGE, highlighting that SDS-PAGE does not preserve protein complexes because it unfolds the proteins.

💡Blue Native PAGE

Blue Native PAGE is a variant of Native PAGE that utilizes Coomassie Blue dye to non-denaturedly bind proteins and protein complexes, imparting a negative charge that allows their movement through a gel during electrophoresis without disrupting their native structures. This technique is particularly useful for analyzing proteins that are not inherently negatively charged at the pH of the gel buffer. The script describes using Blue Native PAGE to visualize proteins while maintaining their complex formations, emphasizing its role in providing a negative charge to proteins without denaturing them.

💡Coomassie Blue dye

Coomassie Blue dye is a staining agent commonly used in biochemical methods for detecting and visualizing proteins. When applied in Blue Native PAGE, it gently binds to proteins, conferring a negative charge to them without disrupting their complex structures or functions. This characteristic makes it ideal for the electrophoretic separation of proteins in their native form, as discussed in the script. The dye's binding facilitates the migration of proteins through the gel by providing the necessary charge for movement.

💡Electrophoresis

Electrophoresis is a laboratory technique used to separate DNA, RNA, or proteins based on their size and charge by applying an electric field across a gel matrix. Molecules migrate through the gel at different rates depending on their charge and size, allowing for their separation and analysis. The script mentions using electricity to move proteins through the gel in both SDS-PAGE and Native PAGE, underscoring electrophoresis's crucial role in these techniques for analyzing protein size and composition.

💡Protein complexes

Protein complexes refer to groups of proteins that interact with each other to perform a specific function or participate in a particular biochemical pathway. These interactions can be transient or stable and are crucial for understanding protein function in a cellular context. Native PAGE, as mentioned in the script, allows for the analysis of these complexes in their intact forms, providing insights into protein-protein interactions and the formation of multimers.

💡Multimers

Multimers are complexes formed by the assembly of multiple copies of a protein or different proteins. They can be homomultimers if made of the same protein or heteromultimers if composed of different proteins. The formation of multimers is essential for many biological processes. The script highlights Native PAGE's ability to detect these multimer formations by keeping protein complexes together, enabling the study of protein interactions.

💡Glycerol

Glycerol is added to protein samples in electrophoresis to increase their density, ensuring they sink into the wells of the gel and do not float out during the procedure. In the script, it is mentioned as a component of the sample preparation for Blue Native PAGE, emphasizing its role in facilitating the proper loading of samples into the gel wells.

💡Voltage

Voltage refers to the electric potential difference applied across the gel during electrophoresis to drive the movement of charged molecules through the matrix. The script discusses adjusting the voltage during the Blue Native PAGE procedure to control the migration speed of proteins and ensure the effective separation and visualization of the protein complexes.

💡Staining

Staining in the context of electrophoresis is the process of applying a dye, such as Coomassie Blue, to the gel after electrophoresis to visualize the separated proteins. Stains bind to proteins, allowing their detection and analysis. The script describes using Coomassie Blue for both imparting a negative charge during Blue Native PAGE and staining the gel post-electrophoresis to visualize the protein bands, highlighting the dye's dual role in the procedure.

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