Collagen Extraction || GATE Biotechnology BT || Biomaterials || Tissue Engineering
Summary
TLDREl video ofrece una explicación detallada de los procedimientos de aislamiento de colágeno, que incluyen preparación de la muestra, extracción, purificación y reorganización de la molécula de colágeno. Se discuten métodos como la precipitación con sales, extracción ácida y el método enzimático, destacando la importancia de la dialisis y la cromatografía de intercambio cationico para purificar el colágeno. Además, se menciona la reconstrucción de la estructura fibrosa del colágeno para restaurar su integridad después de los procesos de extracción.
Takeaways
- 🔬 Hay cuatro pasos principales en el procedimiento de aislamiento de colágeno: preparación de la muestra, extracción de colágeno, purificación y reformación de la molécula de colágeno.
- 🐂 Las tendones son una fuente preferida para el aislamiento de colágeno, ya que son ricas en este.
- ❄️ Se refrigeran las tendones en agua destilada a 4°C durante tres días para minimizar la contaminación no deseada.
- 💧 El agua en la que se sumergen las tendones se cambia dos veces al día para mantener la pureza del colágeno.
- 🔪 Después de la preparación, las fibras de colágeno se cortan en trozos pequeños y se pulverizan para facilitar la extracción.
- 🧂 El método de precipitación con sales es una técnica común para extraer colágeno, donde se utiliza una solución salina para inducir la autoensamblaje de las moléculas de colágeno.
- 🧬 La diálisis es un paso importante en la purificación del colágeno, permitiendo la eliminación de otros componentes no deseados.
- 🌡️ El método de extracción ácida involucra sumergir las tendones en una solución ácida para solubilizar el colágeno, con EDTA como agente antibacteriano.
- 🧪 El método enzimático de aislamiento de colágeno utiliza pepsina para digestión selectiva, ayudando a purificar el colágeno de otras proteínas.
- 🧬 La cromatografía de intercambio cationico es una técnica de purificación adicional que utiliza la carga eléctrica para separar el colágeno de otras proteínas.
- 🔄 La reconstrucción del colágeno es un paso final que ayuda a restaurar la estructura original del colágeno después de los procesos de extracción y purificación.
Q & A
¿Cuáles son los cuatro pasos principales en el procedimiento de aislamiento de colágeno?
-Los cuatro pasos principales en el aislamiento de colágeno son: preparación de la muestra, extracción de colágeno de la muestra, purificación de colágeno de otras proteínas no colágenas y reorganización de la molécula de colágeno.
¿Por qué se prefieren los tendones como fuente para la extracción de colágeno?
-Los tendones son preferidos como fuente de colágeno porque son ricas en este y son más accesibles para su extracción.
¿Cómo se realiza la preparación de la muestra de colágeno?
-La preparación de la muestra de colágeno implica suspender los tendones en agua destilada fría a 4 grados Celsius durante tres días, cambiar el agua dos veces al día, descongelar y lavar los tendones con agua fría, cortar los fibras colágenas en trozos pequeños y pulverizarlos, y luego secarlos durante 24 horas a 40-45 grados Celsius.
¿Qué son los tres métodos importantes para la extracción de colágeno?
-Los tres métodos importantes para la extracción de colágeno son el método de precipitación con sales, el método de extracción ácida y el método enzimático.
¿Cómo funciona el método de precipitación con sales para extraer colágeno?
-El método de precipitación con sales implica aumentar la concentración de sales en la solución para que los colágenos, que son lipófilos, se autoensamblen y se precipiten, rodeados por una capa de agua y sales.
¿Qué es la diálisis y cómo se utiliza en la purificación del colágeno?
-La diálisis es un proceso que utiliza una membrana permeable para separar sustancias disueltas en un líquido. En la purificación del colágeno, se utiliza para eliminar proteínas no deseadas y otros contaminantes, dejando aisladas las proteínas colágenas.
¿Cuál es el propósito de utilizar ADTA en el método de extracción ácida de colágeno?
-El ADTA se utiliza en el método de extracción ácida como agente antibacteriano para evitar el crecimiento de biofilms y como agente de complexación para evitar la presencia de cations divalentes.
¿Qué es el método enzimático de extracción de colágeno y cómo funciona?
-El método enzimático de extracción de colágeno implica tratar los tendones con acetato de sodio y luego agregar pepsina para realizar la digestión enzimática. Esto ayuda a solubilizar las fibras colágenas y a remover las proteínas no deseadas.
¿Qué es la cromatografía de intercambio cationico y cómo se utiliza en la purificación del colágeno?
-La cromatografía de intercambio cationico es un método que utiliza colunas con partículas cargadas negativamente para atraer aminoácidos con carga positiva, permitiendo separar proteínas colágenas positivamente cargadas de otras proteínas no deseadas.
¿Por qué es necesario realizar la reconstrucción de la molécula de colágeno después de la extracción?
-La reconstrucción de la molécula de colágeno es necesaria porque las fibras colágenas pueden estar dañadas o desorganizadas después de los procesos de extracción. Este paso ayuda a restaurar la estructura original del colágeno.
Outlines
🔬 Procedimientos de Isolación de Colágeno
El primer párrafo introduce el tema de la discusión sobre los procedimientos de aislamiento de colágeno, destacando los cuatro pasos principales: preparación de la muestra, extracción de colágeno, purificación y reformación de la molécula de colágeno. Se menciona que las tendones son una fuente preferida para el aislamiento y se describe el proceso de preparación de la muestra, incluyendo la hidratación en agua fría y la limpieza para eliminar impurezas. El colágeno se corta en trozos pequeños y se pulveriza antes de ser secado. Se presentan tres métodos de extracción: precipitación con sales, extracción ácida y método enzimático, y se discute brevemente la teoría detrás de la precipitación con sales.
🧪 Métodos de Extracción de Colágeno
El segundo párrafo se enfoca en los métodos de extracción de colágeno, incluyendo la precipitación con sales, donde el aumento de concentración de sales provoca la autoensamblaje de las moléculas de colágeno. Se describe el proceso detallado de diálisis para purificar el colágeno, eliminando reagentes no deseados. El método de extracción ácida se discute con la utilización de ácido y EDTA para prevenir la formación de biofilms y la generación de cations. El método enzimático se presenta como una alternativa, utilizando ácido acético y pepsina para solubilizar y purificar el colágeno. Al final, se menciona la necesidad de una solución de colágeno puro para propósitos experimentales y se describe el proceso de diálisis para alcanzar esta pureza.
🛠 Purificación y Reconstrucción de Colágeno
El tercer párrafo se centra en la purificación y la reconstrucción de colágeno. Se explica cómo la extracción puede alterar la estructura de las fibras de colágeno, y por lo tanto, es necesario volver a construir su estructura original utilizando metales como calcio o zinc. Se discute el uso de cromatografía de intercambio cationico para purificar el colágeno, aprovechando la carga positiva de las proteínas colagenicas en comparación con otras proteínas no colagenicas. Se describe el uso de carbóximetilcelulosa como un grupo de intercambio negativamente cargado en la cromatografía. Además, se menciona la posibilidad de reconstruir las enlaces cruzados rotos utilizando aldehídos neutros.
Mindmap
Keywords
💡Colágeno
💡Isolemento
💡Extracción
💡Purificación
💡Precipitación con sales
💡Diálisis
💡Método ácido
💡Método enzimático
💡Cromatografía de intercambio cationico
💡Reconstrucción
Highlights
Introduction to the four main steps in collagen isolation procedures.
Explanation of sample preparation, including the selection of collagen sources and initial cleaning process.
Description of the cold distilled water treatment for tendons to remove non-collagenous tissue.
Process of cutting collagen fibers into small pieces and their subsequent drying for further extraction.
Overview of three extraction methods: salt precipitation, acid extraction, and enzymatic isolation.
The theory behind salt precipitation and its role in collagen self-assembly.
Use of dialysis in collagen purification to remove non-collagenous proteins.
Details on the salt precipitation method, including the use of neutral salt solutions and centrifugation.
The role of pH in collagen solubility and its impact on the extraction process.
Process of acid isolation, including the use of acidic solutions and the antibacterial properties of EDTA.
Enzymatic method of collagen isolation, emphasizing the use of pepsin for proteolysis.
The significance of multiple rounds of purification to ensure the purity of collagen.
Technique of salting out to precipitate pure collagen from the supernatant.
The necessity of dissolving precipitated collagen for experimental purposes.
Use of dialysis to remove reagents such as acetic acid and pepsin from the collagen solution.
Extra purification step involving cation exchange chromatography for further refinement of collagen.
The reconstruction of collagen to restore its original fibrous structure after extraction.
Application of metal ions and cross-linking agents in collagen reconstruction.
Principle of cation exchange chromatography and its use in separating collagen from other proteins.
Transcripts
hello guys welcome to my youtube channel
so today we are going to discuss
collagen isolation procedures so there
are mainly four steps in collagen
isolation procedures the first step is
the preparation of the sample second
step is the extraction of collagen from
the sample third step is the
purification of collagen from the other
non collagenous proteins and fourth step
is the reformation of the collagen
molecule so these are basically four
steps in the total isolation procedure
of collagen so first is the preparation
of collagen so first we have to choose a
source from which collagen can be
isolated so generally tendons are the
preferred sources of choice from which
collagen can be isolated so
are tendency in cold distilled water at
four degree celsius for three days so
first they are suspended it playing
water for four degrees celsius at three
days next the water was changed two
times per day because what happens
sometimes we tend on some
non uh tendon thing or the uh something
which we are not interested they also
come out from the tissues so the water
needs to be changed and we are
purely interested in only collagen
nothing other than that so after that
uh so after this water changing so fixed
mass of tendon was defrosted and washed
with cold water several times so why
because
when you defrost or decelerate tendon
what happens the collagen pieces tend to
come out from the tendon so this purpose
of this step is to get collagen from the
tendons next the collagen fibers were
cut into small pieces of one centimeter
in length and pulverized in a mill this
is purely for our
experimental purposes and then they are
dried for 24 hours at 40 to 45 degree
celsius so now we have prepared our
collagen
sample from which we will do further
extraction procedures
so extraction can be performed by three
important procedures either by salt
precipitation method either by acid
extra extraction method or by enzymatic
isolation method so we can use any one
of the procedures to extract pure
collagen all right so first let us
discuss the theory behind salt
extraction so as you can see in any sort
of salt precipitation what happens uh
suppose these are the
blue ones are the collagen molecule here
you have the water molecule and here you
have the salt so when the salt
concentration is much lower collagen and
water will more or less mix with each
other but as you increase the
concentration of salt what happens
collagen molecules they are hydrophobic
in nature so they will
only a self assemble with each other and
they will be surrounded by the
interaction of water and the salt
molecules all right so in this way
gradually collagen molecules will self
assemble with each other and they will
be sounded by a layer of water and salt
so in this way you can get you can
precipitate collagen from the entire
mixture so this is the method basic
method of salting out likewise you have
dialysis of collagen where you can keep
the collagen along with the
media in a dialysis bank so what happens
all the non collagenous proteins and
other proteins which we are not
interested
they will go out into the medium and the
collagen will be pure collagen will
remain in the
back dialysis back after that we can
isolate pure collagen from the dialysis
bank
so generally there are two different
methods there is another third matter so
these two are basically chemical
chemical methods of collagen extraction
so number one is a salt precipitation
method so in salt precipitation method
what happens you create treat tendon
pieces with neutral soil solutions like
0.05 molar na2hpo4
at ph of 8.8 to 9.6 now what is the
reason of treating collagen with this
neutral soil solution the reason is that
purified collagen swells little and
dissolves hardly at ph ranges from 5 to
11 so if you add a little bit of neutral
solution they can dissolve readily in
this ph range so the basics idea of
using neutral source solution is that to
have a good solubility of collagen into
the
isolation medium then they have
centrifuge and for 40 000 to 50 000 g
for one to two hours to remove suspended
particles all the non collagenous
proteins or the extra
uh particles that we are not interested
in they are removed by centrifugation
and the collagen was
isolated from the supernatant then they
are treated with the sodium chloride
solution this here they are performing
salting out method so what will happen
the chloride ions will gradually
surround the collagen molecule and the
collagen molecule will self assemble
and they are using here four molar nacl
at five degrees celsius so whatever they
have got the collagen from the salting
out they are again performing dialysis
all right uh so
why dialysis because you have used uh
some reagents like neutral salt solder
neutral salts in it to hpo4 or nacl so
after you get the pure collagen you need
to remove this extra reagents which you
have used in the isolation procedure so
dialysis is basically
for purification
and to remove the nh2 hpo4 and nacl that
you have used before
and any other reagent then you have the
method of acid isolation so acid
isolation what you do you
soak tendon pieces with 0.5 molar acidic
acid in presence of 5 millimolar adta
now what is the purpose ability is that
purpose abilities it acts as an
antibacterial agent why because it does
not allow
to grow biofilms so when you are
isolating collagen because it is
actually a tissue extract sometimes
whatever bacterial growth might occur
biofilm growth can occur so if you add
an eta that
it will not
let bacteria or biofilms to grow on it
moreover it is also generating agent so
it can also cheat
if present some divalent uh cations also
so the ph is 2.523 which is acidic ph
and for 48 to 96 hours at four day
celsius on four day celsius so under
constant temperature and pressure then
the supernatant
and then you are centrifuging it again
and the supernatant that you are getting
is the acid soluble fraction of the
collagen you are filtering that and the
filtrate was again salted out with nacl
like the previous case you are also
applying salting out method in this case
also
[Music]
next we come to the biological method of
collagen isolation which is the
enzymatic method of collagen isolation
so here what you are doing you are first
performed treating the tissue with 0.05
molar na2hpo4 solution at ph 8.7 to
9.104 degrees celsius to remove all the
non collagenous proteins and then you
are isolating pure tendons only tendons
you are not isolating any other thing
other than the tendon other extra things
have been removed next you are uh
your
next you are
treating this tendon pieces with 0.5
molar acetic acid in presence of 5
millimolar adta so or in presence of
acetic acid vdt what happens from the
tendon the collagen fibers come out so
and then you dissolve that collagen
fibers
in
uh the media and then you add pepsin for
24 hours have four digesters now white
pepsi sometimes uh tendons not only
contain collagen but other
non-collagenous proteins to remove
the other non-collagenous proteins you
have prevention which will actually
perform proteolysis so then this process
is repeated three to four times then
after that it is centrifuge at 5000 g
for 15 minutes and the supernatant is
collected why because in the supernatant
the collagen will be there and in the
pellet other particles will be there
which we will we are not interested in
that
so from the supernatant by salting out
method we are isolating precipitate of
pure collagen so by salting out method
what we are getting precipitate of pure
collagen but we need solution of pure
collagen for experimental purposes so
what we will do we will dissolve it
again in one millimolar acidic acid and
then we are performing dialysis why
because we have used reagents like
acetic acid we have used reagents like
pepsin earlier and
and we don't need this reagents in our
final mixture so we will perform
dialysis with point zero two molar no2
hpo4 solution for 24 hours at 40
resources and finally on dissolving the
precipitate of pure correlation with
acetic acid we get the solution of pure
collagen which was
our
target
[Music]
so
uh after getting solution of pure
collagen we again perform sometimes
purification step this is extra
purification step which we are
performing with the help of cation
exchange chromatography
and after all this purification and
extraction
sometimes reconstruction of collagen is
also done why reconstruction of collagen
is done because the collagen that you
get after acid extraction of salt
extraction that collagen if you see on
the electron microscope you will see
that most of the fibrous are
badly disrupted so after acid extraction
or salt extraction what happens most of
the collagen fibers get
disturbed and they are badly disrupted
so you need to reconstruct that collagen
into the original structure so this step
is purely for that by adding certain
metal lines like l3 plus zn2 plus ca2
plus you can also perform this
reconstruction and if some cross linking
gets broken then you can perform this by
neutral aldehyde
[Music]
now purification how you can do as you
can see in case of cation exchange
chromatography if i talk about the
principle you can use the negatively
charged beads all right so these are the
negatively charged bits and you uh pour
in the mixture of amino acids positively
charged amino acid and negatively
charged amino acids so you pour in the
mixture of positively and negatively
charged amino acids now what will happen
the negatively charged beads will have
attraction for the positively charged
amino acids so the positively charged
amino acid will bind to this negatively
charged
bit column okay so the column contains
negatively charged b so the positively
charged amino acids will get bound to
this
negatively charged bits and the
negatively charged amino acids because
of electrostatic repulsion they will
flow out all right they will not bind
and they will float in this way you can
separate the positively charged
particles from the negatively charged
particles by cation exchange
chromatography
all right so in case of cross collagen
extraction or collagen purification we
use carboxy methyl cellulose
group in the ionized form which in the
ionized form is negatively charged so we
can use it in construction of beads in
the column so and collagen is positively
charged hp is less than seven so if we
design a cation exchange chromatography
where we use
ionized form of carboxymethyl cellulose
as beads and if we then pour the mixture
of collagen and other proteins what will
happen collagen because it is positively
charged will get attracted and stick to
the negatively charged carboxymethyl
group and the other non-collagenous
proteins will flow so in this way we can
purify the collagen
[Music]
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