His tag protein purification | Application of his tag purification | Affinity chromatography
Summary
TLDRThis video script delves into His-tag protein purification, a method utilizing single-step affinity chromatography with a nickel-nitrilotriacetic acid (Ni-NTA) matrix. The technique leverages the specific interaction between the matrix and a hexahistidine tag attached to the protein of interest, enabling efficient separation from a protein mixture. The process involves equilibration, binding, washing, and elution steps, ensuring the purification of the native protein. Advantages include compatibility with cell culture media, ease of use, sensitivity, and cost-effectiveness, though some optimization may be necessary to minimize non-specific binding.
Takeaways
- đ§Ș His-tag protein purification utilizes a single-step affinity chromatography method with immobilized metal ion chromatography (IMAC).
- đ The purification relies on the specific interaction between a nickel-nitrilotriacetic acid (Ni-NTA) matrix and a hexahistidine tag attached to the protein of interest.
- đŹ The hexahistidine tag is incorporated at the DNA level, either at the N-terminal or C-terminal of the open reading frame, and then expressed in bacterial or mammalian cells.
- đŠ The workflow of His-tag purification includes equilibration, binding, washing, and elution steps to separate the tagged protein from a mixture.
- đĄïž Equilibration buffer must be compatible with the protein of interest, considering factors such as ionic strength and pH to maintain protein stability.
- đ During the binding step, the tagged protein binds to the Ni-NTA matrix through non-covalent interactions like coordinate bonds, while other proteins in the lysate do not bind.
- đż The washing step removes non-specifically bound proteins, ensuring that only the specifically bound tagged protein remains on the column.
- đ The elution step involves using a buffer with altered pH and ionic strength to release the bound protein, which can then be collected.
- đ Post-purification, techniques like SDS-PAGE and Western blot can be used to verify the success of the purification process.
- đ° His-tag purification is cost-effective compared to other chromatographic techniques such as gel filtration or HPLC.
- âïž Optimization may be required to minimize non-specific binding, which involves adjusting the ionic strength of the buffers used in the process.
- đ Additional resources and flashcards on this topic can be found on the instructor's social media platforms, with links provided in the video description.
Q & A
What is His-tag protein purification?
-His-tag protein purification is a method that allows for the single-step purification of proteins using immobilized metal ion affinity chromatography with a nickel-nitrilotriacetic acid (Ni-NTA) matrix. The protein is tagged with hexahistidine, which interacts specifically with the matrix.
How is the His-tag attached to the protein?
-The His-tag is attached to the protein by cloning the open reading frame with a hexa-histidine tag at the N-terminal or C-terminal into an expression vector, which is then introduced into bacterial or mammalian cells to produce the tagged protein.
What is the principle behind the separation in His-tag protein purification?
-The principle behind the separation is the specific interaction between the hexahistidine tag on the protein and the nickel ions on the Ni-NTA matrix, which involves non-covalent interactions like coordinate bonds.
What are the steps involved in the His-tag protein purification process?
-The steps are equilibration, binding, washing, and elution. Equilibration involves running a buffer through the column, binding is where the tagged protein attaches to the matrix, washing removes non-specific interactions, and elution collects the purified protein.
Why is the equilibration buffer important in the purification process?
-The equilibration buffer is important because it ensures that the column material is soaked and equilibrated at a specific pH that is compatible with the protein of interest, preventing denaturation.
What factors should be considered when choosing the equilibration buffer?
-Factors to consider include ionic strength and pH, as these can affect protein stability and prevent denaturation during the purification process.
How does the binding step in His-tag purification work?
-In the binding step, the protein sample, usually a cell lysate, is loaded onto the column. The tagged protein binds to the Ni-NTA matrix through non-covalent interactions, specifically coordinate bonds.
What is the purpose of the washing step in the purification process?
-The washing step ensures that any non-specific, untagged proteins are removed from the column, breaking down weak bonds and retaining only the specific interactions between the His-tag and the matrix.
How is the elution step different from the other steps in His-tag protein purification?
-The elution step involves using a buffer with altered pH and ionic strength to loosen the binding between the matrix and the protein, allowing the purified protein to be collected.
What methods can be used to verify the success of His-tag protein purification?
-SDS-PAGE and Western blot can be used to verify the purification by checking the presence and purity of the protein of interest.
What are some benefits of using His-tag protein purification compared to other techniques?
-Benefits include compatibility with cell culture media and lysates, ease of use, sensitivity, specificity, and being more cost-effective compared to techniques like gel filtration chromatography or HPLC.
What are some potential disadvantages of His-tag protein purification?
-Disadvantages include the need for optimization to minimize non-specific binding, which may require calibration of the ionic strength of the equilibration or binding buffer.
Outlines
đŹ His Tag Protein Purification Method
This paragraph introduces His tag protein purification, a single-step affinity chromatography technique for protein separation using a nickel-nitrilotriacetic acid (Ni-NTA) matrix. The process hinges on the specific interaction between the matrix and a hexahistidine tag attached to the protein of interest, facilitated by coordinate bonds. The method begins with cloning a hexa-histidine tag into an expression vector at the protein's N- or C-terminus. Once introduced into a host cell, the vector produces the tagged protein. The purification involves equilibration, binding, washing, and elution steps, with careful consideration of buffer compatibility to maintain protein stability. The technique's advantages include its ability to isolate a specific protein from a mixture and its straightforward workflow. Post-purification, verification is done through SDS-PAGE and western blot.
đ Advantages and Considerations of His Tag Purification
The second paragraph delves into the benefits of His tag protein purification, emphasizing its compatibility with cell culture media and lysates, ease of use, sensitivity, and specificity. It is noted as a cost-effective alternative to other chromatographic techniques such as gel filtration or HPLC. However, the method may require optimization to reduce non-specific binding, which involves adjusting the ionic strength of the equilibration and binding buffers. The paragraph also invites viewers to engage with educational content on the creator's social media platforms, including flashcards, notes, and daily MCQs with prizes, and provides information on how to support the channel through Patreon, the Beam Upi app, or the Super Thanks feature.
Mindmap
Keywords
đĄHis-tag protein purification
đĄAffinity chromatography
đĄHexahistidine tag
đĄExpression vector
đĄEquilibration buffer
đĄBinding
đĄWash buffer
đĄElution
đĄSDS-PAGE
đĄWestern blot
đĄOptimization
Highlights
His tag protein purification is a single-step affinity chromatography method using immobilized metal ion chromatography.
Proteins are tagged with hexahistidine for specific interaction with the nickel NTA matrix.
The hexahistidine tag allows for the separation of the protein of interest from a mixture of proteins.
The expression vector is used to attach the His tag at the DNA level, either N-terminal or C-terminal.
The vector introduction into bacterial or mammalian cells leads to the production of tagged proteins.
The workflow of His tag purification includes equilibration, binding, washing, and elution steps.
Equilibration ensures the column material is soaked and compatible with the protein of interest's pH.
Binding involves the interaction of the tagged protein with the matrix via non-covalent coordinate bonds.
Washing removes non-specifically bound proteins, retaining only the specific interactions.
Elution uses a buffer with altered pH and ionic strength to release the bound protein.
SDS-PAGE and Western blot can be used to verify the success of the purification process.
His tag purification is compatible with cell culture media and lysates, easy to use, sensitive, and specific.
It is a cost-effective alternative to other chromatographic techniques like gel filtration or HPLC.
Optimization may be required to minimize non-specific binding and standardize buffer conditions.
The technique's specificity is a significant advantage for protein purification.
Follow the instructor on social media for flashcards, notes, and daily MCQs on related topics.
Support the channel through Patreon, BeamUpi app, or by clicking the Super Thanks option.
Transcripts
in this video we'll talk about
his tag protein purification
his tag protein purification is a method
by which proteins can be purified by a
single step affinity chromatography by
using immobilized metal ion
chromatography now in this case
the affinity is the key principle behind
separation
in this technique a ni ntm matrix is
used a nickel nta matrix is used
and the protein is tagged with
hexahistidine
and there is an interaction between
these matrix and this particular
histidine tag which is very specific and
there are non-covalent interactions like
coordinate bonds between these two
and that that is how the protein gets
attached with this matrix and it's easy
to separate
so where does the histac come from
actually we have to attach this his tag
with the protein
in order to do so we have to start with
an expression vector where we clone our
open reading frame
and
in the n-terminal or the c-terminal of
this particular protein one can attach a
hexa-histidine tag at the dna level
now once this particular vector is
introduced into the bacterial culture or
let's say mammalian cell which depends
on the type of expression vector we are
using
these expression vector would give rise
to the protein inside these cells
and ultimately these proteins would be
tagged with this hexahistidine
so what is the advantage of this kind of
tag
using this tag we we can understand
where is our protein of interest in a
mixture of other proteins so we can
separate them from a mixture of proteins
now like any column chromatography this
particular
chromatography has a simple workflow
so it starts with a equilibration step
then a binding step where our protein of
interest binds
then a wash and a illusion step
let us go through these steps
in a bit slowly so first step is
equilibration where we run our
equilibration buffer through the column
in this equilibration step the column
material gets soaked and equilibrated
for a particular ph
now the buffer that we have are using it
has to be compatible with the protein of
interest that we want to purify
factors to keep in mind while choosing
this buffer are ionic strength and ph
so factors such as ionic strains are
limited by protein stability so if we
change the ionic strength the protein
might get denatured and while we are
purifying the protein we don't want a
denatured protein right we want it in
its native state
next step is binding here we give the
protein
or the cell lysate
so in this step
there are multiple proteins which are
present in the lysate
but our protein is tagged so it is able
to bind to the matrix now protein
samples are generally dissolved in
specific buffers and loaded into the
columns proteins bind to the column via
several non-covalent interaction in this
case
the non-covalent interaction is
coordinate bonds
once protein have been immobilized in
the stationary phase then we have to use
wash buffer so the next step is washing
washing nco ensures that you don't have
any other non specific bound stuff in
the column all the non-specific untagged
proteins would be washed away in this
stage during the stage of washing all
the
all the weak bonds or non-covalent
interactions are broken down and only
the specific interactions are retained
now last step is illusion in this
illusion step we want to collect the
protein that is bound in the column
so
after all the non-specific interactions
are removed now we are using a
particular buffer which has a altered ph
and ionic strength which would loosen up
the binding between the matrix and the
protein of interest and then it would
come down into the
tube and we can collect that
so now we understand how we can use his
tag protein purification to purify our
protein of interest from a mixture of
protein
once we have purified our protein it is
important to
understand and cross check whether we
have uh whether this assay has worked or
not in order to do that we can run an
sds page and further do a western blot
against this particular protein
anyway let's talk about the benefits of
this purification this is compatible
with cell culture media and lysates
easy to use and quite sensitive and
specific
it is cheaper than many other
chromatographic technique such as gel
filtration chromatography or let's say
other kind of hplc techniques
anyway there are certain disadvantages
but that can be overcome the
disadvantage includes
this technique often requires
optimization to minimize non-specific
binding so many of the cases one has to
calibrate the ionic strength of the
equilibriation buffer or let's say the
binding buffer etc so little bit of
standardization is required but this
technique is really really specific and
important
you can get many flashcards and notes
regarding to these topics in my facebook
page you can follow me on instagram and
my instagram and facebook handle is
provided here links are provided in the
description
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are provided in the description feel
free to connect see you in the next
video
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