Eastern blotting
Summary
TLDRThis video tutorial offers a concise introduction to various blotting techniques in molecular biology, focusing on Southern, Northern, and Western blotting. It explains how these methods are used to identify specific DNA, RNA, and protein sequences. The video also delves into Eastern blotting, a technique for detecting post-translational modifications in proteins, which is crucial for understanding protein function and localization. The tutorial clarifies the differences between blotting techniques and highlights the importance of antibodies in targeting modified regions of proteins.
Takeaways
- 𧬠**Blotting Techniques Overview**: The video introduces various blotting techniques used in molecular biology, including Southern, Northern, Western, and Eastern blotting.
- π **Southern Blotting**: This technique is used for identifying a specific segment of DNA sequence from a gel after electrophoresis.
- π **Transferring DNA**: DNA from the gel is transferred to a nitrocellulose paper, which is easier to handle and binds with DNA.
- π¬ **Probes for Detection**: Probes, either radioactive or fluorescent, are used to bind to the specific DNA region of interest for detection.
- π **Northern Blotting**: Similar to Southern blotting but used for RNA, this technique was developed in 1975.
- π₯ **Western Blotting**: Focused on proteins, this method was developed in 1979 and 1981 to identify specific proteins from a gel.
- π **Eastern Blotting**: Introduced later, this technique is used to detect post-translational modifications (PTMs) in proteins.
- π **Post-Translational Modifications**: PTMs like glycosylation, methylation, and acetylation are crucial for determining the function and location of proteins within a cell.
- π·οΈ **Addressing Proteins**: PTMs act as 'addresses' for proteins, directing them to specific cellular locations, such as mitochondria or outside the cell.
- π οΈ **Eastern Blotting Process**: Involves transferring proteins from an SDS-PAGE gel to a membrane, then using antibodies to detect modified regions of proteins.
Q & A
What is the primary purpose of blotting techniques in molecular biology?
-Blotting techniques are used to identify and detect specific segments of DNA, RNA, or proteins from a gel after electrophoresis.
What are the three main types of blotting techniques mentioned in the script?
-The three main types of blotting techniques mentioned are Southern blotting, Northern blotting, and Western blotting.
What does Southern blotting involve and why is it called so?
-Southern blotting involves the identification of a specific segment of DNA sequence from a gel. It is named after the scientist who developed it, E.M. Southern.
How is Northern blotting different from Southern blotting?
-Northern blotting is used for RNA instead of DNA, and it was named as such to reflect the directionality after Southern blotting.
What is the main focus of Western blotting?
-Western blotting is used to detect specific proteins from a gel, and it involves the use of antibodies to bind to the protein of interest.
What is Eastern blotting and when was it developed?
-Eastern blotting is a technique used to detect post-translational modifications in proteins. It was formulated in 2009 and began to gain attention from then on.
What are the two types of Eastern blotting mentioned in the script?
-The two types of Eastern blotting mentioned are Middle Eastern and Far Eastern blotting.
Why is the production of specific antibodies crucial in Eastern blotting?
-In Eastern blotting, the production of specific antibodies is crucial because they must target the modified regions of proteins, not just any region, to detect post-translational modifications.
How do primary and secondary antibodies differ in the context of blotting techniques?
-Primary antibodies bind directly to the protein of interest, while secondary antibodies bind to the primary antibody and often carry a fluorescent tag for detection.
What is the significance of post-translational modifications in proteins?
-Post-translational modifications are significant because they can alter protein function, stability, and cellular location, acting as 'addresses' for where the protein should be delivered within or outside the cell.
What is the role of GGI bodies in the context of protein modifications mentioned in the script?
-GGI bodies (Golgi apparatus) play a role in post-translational modifications of proteins, where proteins are modified and packaged into vesicles for their final destination within or outside the cell.
Outlines
𧬠Introduction to Blotting Techniques
This paragraph introduces the viewer to various blotting techniques in molecular biology and recombinant DNA technology. The speaker explains that blotting is a method used to identify specific segments of DNA or RNA from a gel. Southern blotting is highlighted as a technique for identifying a specific DNA sequence, while Northern blotting is used for RNA. The process involves transferring DNA from a gel to a nitrocellulose paper, which is easier to handle, and then using probes to bind to the DNA of interest. The probes can be radioactive or fluorescent, depending on the budget. The paragraph also touches on the development of Eastern blotting, which is used to detect post-translational modifications (PTMs) in proteins, such as glycosylation, methylation, and acetylation. These modifications are crucial for determining the final destination of proteins within or outside the cell.
π¬ Eastern Blotting: Detecting Protein Modifications
The second paragraph delves into the specifics of Eastern blotting, a technique developed to detect post-translational modifications in proteins. The process is similar to Western blotting, starting with SDS-PAGE to separate proteins. After transferring the proteins onto a nitrocellulose paper, antibodies are used to target specific modified regions of the proteins. The speaker emphasizes the importance of having antibodies that bind only to the modified sections, distinguishing Eastern blotting from Western blotting, which targets specific amino acid sequences. The paragraph also mentions the types of antibodies used, such as primary and secondary antibodies, with the latter often carrying a fluorescent tag for visualization. The speaker suggests watching a separate video for a deeper understanding of primary and secondary antibodies. The paragraph concludes by encouraging viewers to like, subscribe, and share the video for more educational content.
Mindmap
Keywords
π‘Eastern blotting
π‘Western blotting
π‘Post-translational modifications (PTMs)
π‘Primary antibody
π‘Secondary antibody
π‘SDS-PAGE
π‘Nitr cellulose paper
π‘Fluorescent probes
π‘Radioactive probes
π‘Gel electrophoresis
Highlights
Introduction to Eastern blotting and its significance in molecular biology.
Explanation of different blotting techniques: Southern, Northern, and Western blotting.
Southern blotting for identifying specific DNA sequences.
Northern blotting for RNA detection, developed after Southern blotting.
Western blotting for protein identification, developed in 1979 and 1981.
Eastern blotting for detecting post-translational modifications (PTMs) in proteins.
Importance of PTMs in determining protein function and localization.
The process of Eastern blotting, including SDS-PAGE and transfer to nitrocellulose paper.
Use of antibodies in Eastern blotting to target modified regions of proteins.
Difference between primary and secondary antibodies in blotting techniques.
Challenges in producing antibodies specific to modified protein regions.
The role of the F(ab)' portion of antibodies in binding to specific protein regions.
The key difference between Eastern and Western blotting in targeting modified vs. unmodified protein regions.
The practical application of Eastern blotting in molecular biology research.
Encouragement to subscribe for more educational content on molecular biology.
Transcripts
hello guys in this video tutorial very
quickly I'll be talking about Eastern
blotting you probably heard that name
before and confused about what is it
there are many different types of
blotting techniques that are available
in molecular biology and recombinant DNA
technology lab uh it is mainly the ma
important things are uh the southern
blotting the northern blotting and
western blotting so this is a very basic
uh slide to demonstrate you different
types of blottings that are available
most popular ones Southern blotting and
Northern blotting because Southern
blotting means in all this kind blotting
means the identification of a DNA
specific segment of a DNA sequence uh
from from the gel that we have because
once we are doing we are separating DNA
based on their lens using the
electroforesis process G electrophoresis
technique after the gel electroforesis
we get a gel where we have bands of DNA
based on their length uh in KB or
kilobase pair we take that gel out we
transfer that gel into a nitr cellulous
paper uh because the paper is easy to
handle gel is fragile it can break and
it's very very fragile most of the time
so we take that uh that uh vile I mean
take that natural cellulose paper with
the DNA because DNA will bind with the
natural cellulous paper and then we uh
what we do we just make that DNA single
standard first and then we add some
probe that can specifically bind to the
region of the DNA we want to find and
that is called the blotting we use
certain type of probes like we can use
radioactive probes or we can also use
floresent probes it depends on us what
amount of budget we want to spend
because fluent probes are costlier than
radioactive probes so after the process
is done probing is done then we rest of
the part will be looted and if it's a
radioactive probe we develop a radiogram
where we see the presence of our desired
DNA or na or frin uh but in case of uh
in case of fluoresence probe also the
fluoresence coloration will visualize
the presence of the DNA RNA or protein
so if you do this blotting technique for
DNA it is called Southern blotting
actually it's developed by uh the
scientist Southern that's why according
to his name it is it's nothing to do
with the direction but once it is
developed uh people try that why it's
developed for DNA so we can do that for
RNA also right so try it for RNA it
works and they call it that first one is
Southern so let's make it Northern it's
like a directionality then they go for
Northern blotting for RNA is discovered
again in 1975 itself U Northern blotting
is developed after that Western blotting
is developed in 1979 and 1981 Western
blotting is for proteins the same thing
separation and finding a specific
protein from a blood and gel that is
called the Western blood these are the
three major uh very important blotting
techniques that are available but also
now we have Eastern blotting to find out
this is actually mainly a a version of
Far Western blotting mainly uh side
version Eastern blotting means uh this
is a blotting process for proteins but
to find out ptms post transational
modifications because you know in ukar
there are multiple post transational
modifications are possible Right like
glycosilation methylation acetylation
sumolation so these are the
modifications that are done that are
being done inside those what we
say you
[Music]
know GGI
bodies are the GGI bodies those
modification will be done because ER is
the place where all the proteins are
made from this ER those proteins are
taken inside the GGI where it is
modified and finally GGI is a chamber
which will put the proteins in vesicles
for their Final Destination it could be
the delivery of the protein outside the
cell those will be secretary proteins
like enzymes and minum hormones and many
things in some cases it can be uh
protein to be delivered into a specific
location of Cell itself like inside
mitochondria or inside plased in case of
plants so those post transational
modifications play a vital role for
designating where exactly the protein
will go just like it's if you say this
is a postal system GGI body is a post
office and uh those letters are vesicles
in that case post transation
modification act as the address where
exactly that protein will go right so
the post transation and modification
will act as the address so it's very
very important so we need to detect some
kind of post transation modification
inside the cell because a specific type
of post transation modification carries
a specific message a specific
modification can tell a protein to be
delivered to mitochondria on other hand
a different modification can tell that
protein to be delivered outside the cell
uh the secretary pathway so that's why
the post translation modification
determination is another important
challenge for us and that's why it's
developed it's developed early I mean
very late in 1982 it's formulated but
after that is in 2009 from 2009 Eastern
blotting slowly start to take over and
there are now two types of Eastern
blotting Middle Eastern and Far Eastern
we don't need to go through that part
but just know basics of Eastern blotting
to detect the post transation
modifications in proteins how could you
detect that again the process of
blotting will be the same because
obviously like other proteins Movement
we have SDS page remember because we are
talking about proteins so we have sodium
doicy sulfate polyacryamide gel
electroforesis SDS page so after this
SDS page is done we have the we have the
SDS page gel in our hand and then we can
transfer this SDS page protein to our uh
filter I mean the paper nitr cellulous
paper in this case we use different
kinds of paper
uh available in the market readily so
you transfer it into the paper once it's
transferred to the paper then the rest
of the things will be very very similar
like the Western blotting that means we
use antibody to tack to the protein
because we generally we cannot directly
add a add a fluoresent Dy to a protein
because a protein have a specific region
to bind so we develop certain antibody
which has the binding section to that
interest protein of our interest so that
that antibody can direct Bine now that
antibody can itself carry a that
antibody is called primary antibody now
that can carry a floresent tag right or
that may not carry a floresent tag we
need to have another secondary antibody
to be B bound with that which carries
the floresent tag in either way either a
single antibody which will detect the
protein may also have the floresent tag
may also carry the floresent tag in that
case we need only one antibody for the
reaction to detect the specific region
of modification or in other case we may
need two different antibodies first
antibody is simply bind to our desired
protein second antibody will bind to the
first antibod that will contain the tag
right uh if you are confused about
primary and secondary antibody I
recommend you to watch my video on
primary and secondary antibody you'll
find a different video completely
different separate video on primary and
secondary antibody you can watch that
video in the YouTube channel just search
it and you can find it and learn it but
the idea here the difference between the
B basic Western blotting and Eastern
blotting is that in this case of Eastern
blotting we
use uh the proteins and the antibodies
that we use they are targeted towards
the modification sites of proteins not
rest of the region of the protein
because in in Western blotting we target
specific sections of amino acid of the
protein but in this case we only target
modified amino acid of the protein
modified regions of the protein for
example a protein for example let's say
protein may have uh let's say
phosphorilation not phosphor let's say
may have a methylation out there now we
develop an
antibody that will tag or that will bind
to the methylated domain of that protein
only it will not bind any other place of
the protein remember this is very very
important so to design and find the
specific antibody which will only bind
with the modified section of the protein
is the key difference between eastern
blotting and western blotting okay rest
of the things are the same so that is
the thing that is the channel challenge
we need to find and we need to figure
out production of that antibody and
actually we can produce different types
of antibody if we know the sequence of
the protein we need to bind if we can
figure that out we can make a specific
antibody specific against that region of
the protein in the faab portion of that
antibody right because faab portion is
the portion to bind with uh the specific
proteins so you can prepare it any time
if you want right so this is the process
of Eastern blotting and that's how the
easn blotting is done very very simple
just like any other blotting but here
the proteins and the antibody that we
target will Target against the modified
region only okay so that's it guys if
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