The main purpose of these slides is to convey information to the Professors, Lecturers, and Students. These slides contain authentic information about this topic which is mentioned in that.
Following is my journal documentation during Master's in Biotechnology completed in 2015. I do understand many changes would've occurred in the curriculum since then, but the basics seldom change. Kindly absorb as per your need.
We are engaged in manufacturing, supplying and exporting of Laboratory Filtration Products (syringe filters, membrane filters, glass fiber filters and filter papers). These filtration products are precisely manufactured by making use of superior quality raw material and ultra-modern techniques by the experience team. Our offered products are widely used in various laboratories for various filtration works in worldwide. http://www.axivasichem.com/syringe-filters.aspx
Following is my journal documentation during Master's in Biotechnology completed in 2015. I do understand many changes would've occurred in the curriculum since then, but the basics seldom change. Kindly absorb as per your need.
We are engaged in manufacturing, supplying and exporting of Laboratory Filtration Products (syringe filters, membrane filters, glass fiber filters and filter papers). These filtration products are precisely manufactured by making use of superior quality raw material and ultra-modern techniques by the experience team. Our offered products are widely used in various laboratories for various filtration works in worldwide. http://www.axivasichem.com/syringe-filters.aspx
BLO: Transferring the macromolecule from gel to membrane followed by detection on the membrane using antibody is k/a blotting
molecular methods used to identify and measure specific DNA, RNA and protein in complex biological mixtures.
It is the technique för
transferring DNA, RNA and proteins onto a carrier so they can be separated, and often follows the use of a gel electrophoresis.
IMMUNO BLOTTING:
Immunoblotting techniques use antibodies to identify target proteins .
They involve identification of protein target via antigen-antibody (or protein-ligand) specific reactions.
The Southern blot is used for transferring DNA,.
The Northern blot for RNA
The western blot for PROTEIN.
The Eastern blot for PROTEIN, post-translational modifications (PTMS) .
WESTERN BLOTTING:
Principle:
Western blotting technique is used for identification of particular protein from the mixture of protein.
In this method labelled antibody against particular protein is used identify the desired protein, so it is a specific test.
Western blotting is also known as immunoblotting because it uses antibodies to detect the protein.
METHODOLOGY:
Extraction of protein
2. Gel electrophoresis: SDS PAGE
3. Blotting: electrical or capillary blotting
4. Blocking: BSA
5. Treatment with primary antibody
6. Treatment with secondary antibody( enzyme labelled anti Ab)
7. Treatment with specific substrate; if enzyme is alkaline phosphatase, substrate is p-nitro phenyl phosphate which give color.
Immobilization of proteins on the solid support of nitrocellulose membrane or polyvinylidinefluoride membrane. Then antibodies bind speciffcally that can be analyzed through Autoradiography
Western blot is a commonly used method for protein analysis. It can be used for qualitative and semi-quantitative protein analysis. For the accomplishment of the western blot, there are three elements, separation of proteins by size, transferring proteins to a solid support, and marking proteins by primary and secondary antibodies for visualization.
Antibody Coupling - Single Coupling Approach To Bind Antibodies Diverse Speci...Anteo Technologies
Traditional covalent chemistries use harsh chemicals and require expertise in the techniques of diverse covalent coupling methodologies. The Antibody Coupling Kit was made to address issues such as: difficulties in binding certain antibodies with traditional covalent chemistries, antibody wastage, and incorrect antibody orientation. Anteo’s technology offers scientists the flexibility to bind any antibody onto a solid support surface through the use of polymeric metal complexes. The polymeric metal nature of the technology allows multi-valent binding of the target antibody through chelation to the electron donating groups located in the Fc region of the antibody. Anteo’s kit promotes gentle monolayer binding, meaning proteins assemble in the correct orientation while reducing the amount of damaged proteins, leading to increased functionality of antibodies and less antibodies used for the experiment.
This application note demonstrates the ability of the Antibody Coupling Kit to bind Mouse IgG, Mouse IgM, Rabbit IgG, Human IgG and Human IgM antibodies onto 200 nm magnetic particles using a particle-based fluorescent antibody loading assay.
BLO: Transferring the macromolecule from gel to membrane followed by detection on the membrane using antibody is k/a blotting
molecular methods used to identify and measure specific DNA, RNA and protein in complex biological mixtures.
It is the technique för
transferring DNA, RNA and proteins onto a carrier so they can be separated, and often follows the use of a gel electrophoresis.
IMMUNO BLOTTING:
Immunoblotting techniques use antibodies to identify target proteins .
They involve identification of protein target via antigen-antibody (or protein-ligand) specific reactions.
The Southern blot is used for transferring DNA,.
The Northern blot for RNA
The western blot for PROTEIN.
The Eastern blot for PROTEIN, post-translational modifications (PTMS) .
WESTERN BLOTTING:
Principle:
Western blotting technique is used for identification of particular protein from the mixture of protein.
In this method labelled antibody against particular protein is used identify the desired protein, so it is a specific test.
Western blotting is also known as immunoblotting because it uses antibodies to detect the protein.
METHODOLOGY:
Extraction of protein
2. Gel electrophoresis: SDS PAGE
3. Blotting: electrical or capillary blotting
4. Blocking: BSA
5. Treatment with primary antibody
6. Treatment with secondary antibody( enzyme labelled anti Ab)
7. Treatment with specific substrate; if enzyme is alkaline phosphatase, substrate is p-nitro phenyl phosphate which give color.
Immobilization of proteins on the solid support of nitrocellulose membrane or polyvinylidinefluoride membrane. Then antibodies bind speciffcally that can be analyzed through Autoradiography
Western blot is a commonly used method for protein analysis. It can be used for qualitative and semi-quantitative protein analysis. For the accomplishment of the western blot, there are three elements, separation of proteins by size, transferring proteins to a solid support, and marking proteins by primary and secondary antibodies for visualization.
Antibody Coupling - Single Coupling Approach To Bind Antibodies Diverse Speci...Anteo Technologies
Traditional covalent chemistries use harsh chemicals and require expertise in the techniques of diverse covalent coupling methodologies. The Antibody Coupling Kit was made to address issues such as: difficulties in binding certain antibodies with traditional covalent chemistries, antibody wastage, and incorrect antibody orientation. Anteo’s technology offers scientists the flexibility to bind any antibody onto a solid support surface through the use of polymeric metal complexes. The polymeric metal nature of the technology allows multi-valent binding of the target antibody through chelation to the electron donating groups located in the Fc region of the antibody. Anteo’s kit promotes gentle monolayer binding, meaning proteins assemble in the correct orientation while reducing the amount of damaged proteins, leading to increased functionality of antibodies and less antibodies used for the experiment.
This application note demonstrates the ability of the Antibody Coupling Kit to bind Mouse IgG, Mouse IgM, Rabbit IgG, Human IgG and Human IgM antibodies onto 200 nm magnetic particles using a particle-based fluorescent antibody loading assay.
Instructions for Submissions thorugh G- Classroom.pptxJheel Barad
This presentation provides a briefing on how to upload submissions and documents in Google Classroom. It was prepared as part of an orientation for new Sainik School in-service teacher trainees. As a training officer, my goal is to ensure that you are comfortable and proficient with this essential tool for managing assignments and fostering student engagement.
How to Make a Field invisible in Odoo 17Celine George
It is possible to hide or invisible some fields in odoo. Commonly using “invisible” attribute in the field definition to invisible the fields. This slide will show how to make a field invisible in odoo 17.
Unit 8 - Information and Communication Technology (Paper I).pdfThiyagu K
This slides describes the basic concepts of ICT, basics of Email, Emerging Technology and Digital Initiatives in Education. This presentations aligns with the UGC Paper I syllabus.
This is a presentation by Dada Robert in a Your Skill Boost masterclass organised by the Excellence Foundation for South Sudan (EFSS) on Saturday, the 25th and Sunday, the 26th of May 2024.
He discussed the concept of quality improvement, emphasizing its applicability to various aspects of life, including personal, project, and program improvements. He defined quality as doing the right thing at the right time in the right way to achieve the best possible results and discussed the concept of the "gap" between what we know and what we do, and how this gap represents the areas we need to improve. He explained the scientific approach to quality improvement, which involves systematic performance analysis, testing and learning, and implementing change ideas. He also highlighted the importance of client focus and a team approach to quality improvement.
The Art Pastor's Guide to Sabbath | Steve ThomasonSteve Thomason
What is the purpose of the Sabbath Law in the Torah. It is interesting to compare how the context of the law shifts from Exodus to Deuteronomy. Who gets to rest, and why?
The French Revolution, which began in 1789, was a period of radical social and political upheaval in France. It marked the decline of absolute monarchies, the rise of secular and democratic republics, and the eventual rise of Napoleon Bonaparte. This revolutionary period is crucial in understanding the transition from feudalism to modernity in Europe.
For more information, visit-www.vavaclasses.com
How to Split Bills in the Odoo 17 POS ModuleCeline George
Bills have a main role in point of sale procedure. It will help to track sales, handling payments and giving receipts to customers. Bill splitting also has an important role in POS. For example, If some friends come together for dinner and if they want to divide the bill then it is possible by POS bill splitting. This slide will show how to split bills in odoo 17 POS.
2. To separate the proteins through SDS-PAGE
and detection followed by characterization of
proteins through Western blotting.
Introduction :
Western blotting (also known as protein- or
immunoblotting) is a rapid and sensitive assay for
detection and characterization of proteins. It works
by exploiting the specificity inherent in Ag-Ab
recognition. It is used to identify specific antigens
recognized by polyclonal or monoclonal antibodies.
Western blotting is carried out along with protein
(antigen) separation in gel by electrophoresis and
the blot development.
3. It is essentially a combination of 3
techniques:
Electrophoresis (PAGE)
Western blotting
Immunochemical detection.
Principle :
Identification of protein separated by gel
electrophoresis is limited by the small pore size of
the gel, as the macromolecule probe for protein
analysis cannot permeate the gel. This limitation is
overcome by blotting the protein into an adsorbent
porous membrane.
4. The apparatus consists of a tank containing buffer,
in which is located a cassette. Clamping the gel
and the membrane tightly together, a current is
applied from electrodes, and repeated on either
side of the cassette to avoid heating effects.
The proteins are separated according to their
electrophoresis mobility and blotted onto the
membrane identified, using suitable
immunochemicals to locate the protein of interest.
The individual techniques are explained below.
5. Stage 1. Prepare a PAGE gel slab and fix to a vertical
electrophoretic apparatus. Treat the sample with suitable
buffer and load onto the gel slots.
Stage 2. Apply electric current. After a few minutes, proteins
in the sample migrate according to their electrophoretic
mobility in the stacking gel. The stacking gel has a
polyacrylamide concentration resulting in higher pore size and
a lower pH of 7. This enables the protein to concentrate into
sharp bands due to isotachopharesis, or band-sharpening
effect. At the end of the stacking gel, it meets the separating
gel, which has a higher polyacrylamide concentration and
higher pH. In the separating gel, the proteins travel according
to their size.
6. Stage 3. When the dye front reaches the bottom of the
separating gel, the proteins in the sample are resolved
depending on their size. However the protein cannot be
visualized directly. The gel needs to be stained with suitable
stainer to visualize all the proteins. The identification of
protein of interest can be done using a suitable probe and a
developing system.
7. Blotting is the transfer of resolved proteins from the gel to
surface of suitable membrane. This is done commonly by
electrophoresis (known as electro blotting).
In this method, the transfer buffer has a low ionic strength
which allows electro transfer of proteins. Methane in the
buffer increases binding of proteins to nitrocellulose and
reduces gel smelling during transfer.
The use of the membrane as a support for protein enables
the case of manipulation efficient washing and faster
reactions during the immuno detection, as proteins are more
accessible for reaction.
(a) The membrane is in close contact with PAGE gel
containing proteins. The proteins are electro transferred to
nitrocellulose membrane.
8. (b) At the end of electro transfer, all proteins would have
migrated to the NC membrane.
The protein was transferred to the corresponding position on
the membrane as on the gel. A mirror image of the gel was
formed. However, the protein location and detection can only
be assessed after immuno detection.
Immunodetection :
The transferred proteins are bound to the surface of NC
membrane and are accessible for reaction with
immunochemical reagents. All the unoccupied sites on the
membrane are blocked with inert proteins, detergents, or
other suitable blocking agents. The membrane is then probed
with a primary antibody and a suitable substrate so the
enzyme identifies the ag-ab complex form on the membrane.
Applications :
To characterize proteins and to identify specific antigens for
antibodies.
9. Blotting Buffer. Add 25 ml of blotting buffer component A
and 25 ml of component B to 150 ml of distilled water.
Other Buffers. Dilute the required amount of buffer
concentrate to 1X concentration with water.
Antibody. Dilute primary A-b and label secondary HRP
conjugate in an assay buffer.
Substrate. Dilute TMB/H2O2, 10X concentration 10 times with
heat just before use.
10. Run SDS: Poly-acrylamide gel electrophoresis.
Electroblot.
Assemble the blotting sandwich within the blotting cassette.
Care should be taken to avoid air bubbles between the gel
and NC membrane.
Insert the cassette into the apparatus filled with blotting
buffer so that the gel faces the cathode.
Connect the power supply and use a voltage of 50 V for 5
hours for blotting.
Immuno-detection :
(a) Remove the NC membrane gently from the cassette and
place it in the blocking buffer for 2 hours at room
temperature, or overnight in the cold.
(b) Suspend the primary antibody in 10 ml with the assay
buffer, using a suitable tube.
11. (c) Immerse the blot in the 10 Ab solute and gently agitate for
30 minutes.
(d) Wash the blot by immersing it in wash buffer for 3–5
minutes. Repeat 2 more times.
(e) Prepare 1:1000 dilutions of labeled 20 Ab in the assay
buffer. Prepare sufficient (10 mL) volume of diluted Ab to
cover the blot.
(f) Immerse the blot in 20 Ab solute and agitate gently for 30
minutes.
(g) Wash the blot in wash buffer for 3–5 minutes and repeat 4
times.
(h) Immerse the washed blot in 10 mL of substrate solution
with gentle shaking. Bands will develop sufficient color within
5–10 min.
(i) Remove the blot and wash with distilled water. Dry.
(j) Although the colored hands fade with time, the rate of
color loss can be retarded if the blots are kept in the dark.
12. The proteins separated through the SDS-PAGE have
been successfully transferred onto the
nitrocellulose membrane and the transferred
proteins detected by immunodetection, which was
confirmed by the development of color bands on
the nitrocellulose membrane.
