SDS PAGE : How does it works?
Summary
TLDRIn this video, the host explains SDS-PAGE (SDS polyacrylamide gel electrophoresis) in under five minutes. SDS-PAGE is a technique used to separate proteins based on their molecular weight. The video covers the difference between stacking and resolving gels, the role of SDS and beta-mercaptoethanol in denaturing proteins, and how proteins are separated using electrophoresis. The process involves various components like chloride and glycine ions. Finally, the host mentions protein staining methods and points viewers to a related video on Western blotting for further analysis.
Takeaways
- 🧬 SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) is a technique used to separate proteins based on their molecular weight.
- 🔬 The gel used in SDS-PAGE consists of a stacking gel and a resolving gel, each with different pH levels and pore sizes to facilitate protein separation.
- 🌡️ The stacking gel has a pH of 6.8 and larger pores with lower ionic strength, while the resolving gel has a pH of 8.8 with smaller pores and higher ionic strength.
- 🧪 Polyacrylamide gels are made from acrylamide and bisacrylamide, and their ratio can be adjusted to create different pore sizes for protein separation.
- 🔄 Proteins are denatured with SDS and beta-mercaptoethanol to break disulfide bonds and mask charges, resulting in uniform negatively charged proteins.
- 🚫 The electrophoresis process involves the movement of proteins from the negative to the positive terminal due to their negative charge from SDS coating.
- 🏃♂️ Small ions like chloride move quickly towards the positive terminal, followed by proteins, with larger, slower glycine ions lagging behind.
- 🏁 Proteins are 'stacked' at the interface of the stacking and resolving gels before being separated in the resolving gel based on their molecular weight.
- 🖼️ After electrophoresis, gels can be stained with Coomassie Brilliant Blue or silver stain to visualize protein bands for analysis.
- 🔍 Western blotting is a further technique used to confirm the presence of a protein of interest in a sample after SDS-PAGE separation.
Q & A
What is SDS-PAGE?
-SDS-PAGE, or SDS polyacrylamide gel electrophoresis, is a technique used to separate proteins based on their molecular weight by utilizing a gel system.
What are the two types of gels used in SDS-PAGE?
-The two types of gels used in SDS-PAGE are the stacking gel, with a pH of 6.8, and the resolving gel, with a pH of 8.8.
Why is there a pH difference between the stacking gel and the resolving gel?
-The pH difference ensures proper separation of proteins. The stacking gel's lower pH helps to 'stack' the proteins at the boundary, while the resolving gel's higher pH facilitates their separation based on molecular weight.
What role does pore size play in protein separation in SDS-PAGE?
-The pore size, which depends on the concentration of acrylamide and bis-acrylamide, affects the separation of proteins. Smaller proteins require higher concentrations of gel to be properly separated.
Why is SDS and beta-mercaptoethanol added to the protein solution?
-SDS denatures proteins and masks their charges with a uniform negative charge, while beta-mercaptoethanol breaks disulfide bonds, ensuring proteins are fully denatured and linearized.
How do proteins move during electrophoresis in SDS-PAGE?
-Since SDS coats proteins with a negative charge, they move towards the positive terminal during electrophoresis, allowing separation based on their molecular weight.
What is the role of chloride ions and glycine in the running buffer?
-Chloride ions, being small and fast, lead the proteins in the electrophoretic process, while glycine ions, which move slowly at pH 6.8, lag behind, aiding in the stacking of proteins in the stacking gel.
How are proteins separated in the resolving gel?
-Once proteins reach the resolving gel, they are separated based on their molecular weight, with larger proteins moving slower than smaller ones.
What staining techniques can be used to visualize proteins after SDS-PAGE?
-Proteins can be stained using techniques like Coomassie Brilliant Blue staining or silver staining to visualize the separated proteins on the gel.
What additional step is required to confirm the presence of a specific protein in the sample?
-To confirm the presence of a specific protein, a Western blot is performed after SDS-PAGE, where the protein of interest is detected using specific antibodies.
Outlines
🧬 Understanding SDS-PAGE: Protein Separation Technique
This paragraph introduces the concept of SDS-polyacrylamide gel electrophoresis (SDS-PAGE), a technique used to separate proteins based on their molecular weight. The video explains the gel-based separation process involving two types of gels: the stacking gel and the resolving gel. The stacking gel has a pH of 6.8 and larger pores with lower ionic strength, while the resolving gel has a pH of 8.8, smaller pores, and higher ionic strength. The percentage of polyacrylamide and bisacrylamide in the gels can be varied to create different pore sizes, which is crucial for separating proteins of different sizes. The paragraph also discusses the use of SDS and beta-mercaptoethanol to denature proteins, remove disulfide bonds, and mask charges, resulting in a uniform negative charge. This preparation allows proteins to be separated based on their size during electrophoresis.
🔬 SDS-PAGE Electrophoresis Process and Visualization
This paragraph delves into the electrophoresis process of SDS-PAGE, where negatively charged proteins move towards the positive terminal. The movement of chloride ions, which are small and move quickly, is contrasted with the bulkier proteins that move more slowly. The paragraph explains the role of glycine ions at pH 6.8, which behave as cations and move slowly, contributing to the stacking of proteins at the top of the resolving gel. This stacking effect aligns proteins at the starting line, allowing them to be separated based on their molecular weight. The video concludes with a discussion on visualizing the results of the electrophoresis through staining methods like Coomassie brilliant blue or silver staining. It also mentions the use of Western blot for further analysis of specific proteins of interest, with a call to action for viewers to watch a related video on Western blotting.
Mindmap
Keywords
💡SDS-PAGE
💡Polyacrylamide Gel
💡Stacking Gel
💡Resolving Gel
💡Molecular Weight
💡SDS
💡Beta Mercaptoethanol
💡Running Buffer
💡Glycine
💡Western Blot
Highlights
Introduction to HTS-PAGE, a technique for separating proteins by molecular weight.
Explanation of the gel-based separation system using a combination of running and resolving gels.
Difference in pH between stacking gel (6.8) and resolving gel (8.8) and its significance.
Varying percentages of resolving gel to achieve different pore sizes for protein separation.
Composition of polyacrylamide gel made from acrylamide and bisacrylamide.
Adjustable ratio of acrylamide to bisacrylamide to create different pore sizes in the gel.
Use of HTS and beta mercaptoethanol to denature proteins and mask charges.
Impact of SDS treatment on protein structure, leading to uniform charge and linearization.
Loading of protein samples onto the gel for electrophoresis.
Movement of negatively charged proteins from negative to positive terminal during electrophoresis.
Role of chloride ions as leaders in the electrophoretic process due to their small size.
Glycine ions' behavior at pH 6.8 and their slow movement in the gel.
Protein stacking at the resolving gel interface, preparing for molecular weight-based separation.
Proteins' movement and separation in the resolving gel based on their molecular weight.
Completion of gel running, visualization of the unstained gel, and subsequent staining methods.
Use of Coomassie Brilliant Blue or silver staining for visualizing protein bands on the gel.
Necessity of Western blot for identifying a protein of interest in the sample.
Encouragement to watch the Western blot video for further understanding.
Closing remarks, appreciation for watching, and call to action for likes, shares, and subscriptions.
Transcripts
hi and welcome to my video series of
piyo techniques explained in five
minutes where I explained the concept in
biology in less than 5 minutes or so so
if you haven't yet subscribed to my
channel
hit that subscribe button right now so
today's video is about HTS page so SDS
page or SDS polyacrylamide gel
electrophoresis it's a technique by
which we can separate proteins according
to their molecular weight now there are
this system is based on gel based
separation and the jail is a complex of
two type of jails are running jail and
resolving jail and using a combination
of running gel and the resolving gel
which we would be discussing in this
video one can finally resolve the
proteins according to their molecular
weight so let's go step by step and
understand this process process in
details so here imagine this is the jail
that you need to run for the
polyacrylamide gel electrophoresis it
has two basic region one is Technogel
that is on the top where you load the
samples and here is something called
resolving gel so what is different
between these two region the stacking
gel has a pH of 6.8 and the loading J
and the resolving gel has a pH of 8.8
and why there is a pH difference we
would get to know very soon also the
percentage of these gels are very
different the percentage of resolving
gel varies across the usage of the user
they can use different percentage of
resolving gel and different percent of
resolving gel has different pore size
which would allow them to separate the
samples properly so generally the
stacking gel has larger pore and lower
ionic strength whereas the resolving gel
has smaller pores and higher ionic
strength why is so we would get to know
soon so the poly acrylamide which is
used the poly acrylamide gel which is
used to separate proteins is basically
made up of two components
acrylamide and miss acrylamide so they
polymerize to form this whole shell-like
structure and the percentage of these
poly acrylamide and miss acrylamide can
be changed and their ratio could be
varied
to get different type of pore size and
this pore size difference is important
because let's say we need to separate a
protein which is very small in size so
we would prefer using a higher
concentration of jail right because
protein size could be different and in
order to resolve them we need proper
size now let's just try to start this
process by let's say we have a protein
solution then we add into the protein
solution
HTS and Peter mercaptoethanol and why an
HTS because SDS and beta mercaptoethanol
would first of all denature the protein
get rid of all the disulfide bonds and
kind of mask all of different charges on
the protein by negative charge so after
that you put it in incubator to boil it
for some time so this is the situation
of the protein before a seediest
treatment after SDS treatment you can
see the charge are uniformly Mark's mask
by the SDS and everything is like
uniform and linearized so all the
secondary structures are broken after
this treatment now you would load this
gel and start the gel for and try to
separate the proteins according to the
electro phoretic principle now the
proteins are now negatively charged
because their SDS coated so they would
move from negative to the positive
terminal but there are multiple things
in the solution then use running buffer
one of the component of the running
buffer is chloride ion so chloride ion
is a small ion it would quickly go to
the positive and so it's the leader so
it runs very fast after that the protein
results pretty much slowly because the
protein is big bulkier but it has also
negative charge so it would be attracted
to the positive terminal it would move
towards the resolving gel side and at
the end there is a glycine ion now at pH
6.8 glycine and behave like a cation and
since it's a cation
it's mobility is ultra slow and it is
it is lagged behind in this whole
process so they move very slowly now
let's say they're moving and at the end
of this process what would be happening
is the proteins would be stacked on top
of the resolving gel as if they are like
making their position in the starting
line and from that they would be
resolved according to the function of
their molecular weight right so the pH
of the running gel is closer to the pKa
of glycine that is why it becomes also
negatively charged and it quickly moves
just like the chlorine ion and now the
protein is free and it would move like
its own pace and it would be separated
by its own molecular weight so after
that let's say we stop the timer when
the gel running is complete because we
can understand by the die front and then
this is how our unstained gel look like
we can stain the gel by different method
one such method is using Kumasi
brilliant blue or sometimes silver
staining or anything so you can stain
the gel and you know the expected band
size so you can ultimately check the
stain gel and check the known protein
markers to understand that whether your
protein of interest is there whether the
resolving is happening but in order to
understand a particular protein of
interest is present in your protein
sample or not you have to do Western
blot and if you haven't yet washed my
Western blot video quickly watch the
Western blot and the link is in the
description and thanks for listening I
hope you enjoyed this video if you liked
this video give it a quick thumbs up and
don't forget to Like share and subscribe
thank you
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