The document discusses the precipitin reaction, which involves an antibody binding to its antigen to form an insoluble complex. It precipitates out of solution, forming a visible precipitate. The optimal mutual proportions (OMP) of antigen and antibody yield the greatest amount of precipitation. To determine the OMP, serial dilutions of the antigen are made while keeping the antibody concentration constant. The dilution that produces the maximum amount of precipitation at the OMP. Experiments are described to perform precipitin reactions, analyze the results, and calculate values like the antibody concentration in the original antiserum.
A real-time polymerase chain reaction is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR, i.e. in real-time, and not at its end, as in conventional PCR.
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A real-time polymerase chain reaction is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR, i.e. in real-time, and not at its end, as in conventional PCR.
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This method is used to visualise the localisation and quantity of a protein of interest. The target protein is bound to by a specific primary antibody, which in turn is detected by a secondary antibody conjugated to a fluorophore. A fluorescent or confocal microscope is used to visualise the protein.
Immunocytochemistry (ICC) differs from immunohistochemistry (IHC) in that the former is performed on samples of intact cells that have had most, if not all, of their surrounding extracellular matrix removed. In contrast, immunohistochemical samples are sections of biological tissue, where each cell is surrounded by tissue architecture and other cells normally found in the intact tissue. These differences cause the samples to be prepared differently. For ICC, the sample requires permeabilisation so that the antibodies can reach the intracellular targets. Depending on the thickness of the sample, IHC samples do not require this.
Do you have a technical question? Get in touch: info@stjohnslabs.com
Spectrophotometric methods for determoination of Proteins Sabahat Ali
Different types of assay are present of determination of proteins
Bicinchoninic acid assay, Biuret protein assay, Lowry test assay, Bradford protein assay & Warburg Christion (A280/A260)
Immunodiffusion -Different Types,Principle,procedureand application. it is a diagnostic technique for the detection or measurements of antibodies and antigens by their precipitation which involves diffusion through a substances such as agar or gel agarose .common types -oudin procedure,oakley fulthorpe procedure ,mancini technique ,ouchterlony double immuno diffusion
This lectureis about DNA extraction from whole Blood presented by Tuba nafees she is msc graduate in Biotechnology from University of Karachi, Sindh Pakistan.
lecture video is also there in youtube link:
https://www.youtube.com/watch?v=cGr__SuqYgY&t=409s
Techniques of DNA Extraction, Purification and QuantificationBHUMI GAMETI
Introduction
The overall process…
Uses of isolated genomic DNA
Extraction of DNA from plant material
Components of DNA extraction solutions
Cell Lysis or Cell disruption :
Purification of DNA
CTAB Method
Phenol–chloroform extraction
PROTEINASE K
Salting out
Silica adsorption method
Magnetic beads
FTA Paper
Nucleic acid quantification
Agarose Gel Electrophoresis
UV spectroscopy
DNA quantification using NanoDrop
This method is used to visualise the localisation and quantity of a protein of interest. The target protein is bound to by a specific primary antibody, which in turn is detected by a secondary antibody conjugated to a fluorophore. A fluorescent or confocal microscope is used to visualise the protein.
Immunocytochemistry (ICC) differs from immunohistochemistry (IHC) in that the former is performed on samples of intact cells that have had most, if not all, of their surrounding extracellular matrix removed. In contrast, immunohistochemical samples are sections of biological tissue, where each cell is surrounded by tissue architecture and other cells normally found in the intact tissue. These differences cause the samples to be prepared differently. For ICC, the sample requires permeabilisation so that the antibodies can reach the intracellular targets. Depending on the thickness of the sample, IHC samples do not require this.
Do you have a technical question? Get in touch: info@stjohnslabs.com
Spectrophotometric methods for determoination of Proteins Sabahat Ali
Different types of assay are present of determination of proteins
Bicinchoninic acid assay, Biuret protein assay, Lowry test assay, Bradford protein assay & Warburg Christion (A280/A260)
Immunodiffusion -Different Types,Principle,procedureand application. it is a diagnostic technique for the detection or measurements of antibodies and antigens by their precipitation which involves diffusion through a substances such as agar or gel agarose .common types -oudin procedure,oakley fulthorpe procedure ,mancini technique ,ouchterlony double immuno diffusion
This lectureis about DNA extraction from whole Blood presented by Tuba nafees she is msc graduate in Biotechnology from University of Karachi, Sindh Pakistan.
lecture video is also there in youtube link:
https://www.youtube.com/watch?v=cGr__SuqYgY&t=409s
Techniques of DNA Extraction, Purification and QuantificationBHUMI GAMETI
Introduction
The overall process…
Uses of isolated genomic DNA
Extraction of DNA from plant material
Components of DNA extraction solutions
Cell Lysis or Cell disruption :
Purification of DNA
CTAB Method
Phenol–chloroform extraction
PROTEINASE K
Salting out
Silica adsorption method
Magnetic beads
FTA Paper
Nucleic acid quantification
Agarose Gel Electrophoresis
UV spectroscopy
DNA quantification using NanoDrop
Pet's have allergies. In fact, pets can be allergic to many of the same things that people are. While we sneeze, get watery eyes, or get stuffy; pets are more likely to itch, get hives, or have GI issues. This presentation goes over what an allergy actually is, different ways to test for allergy, and our approach to allergy treatment or long term allergy management.
Diagnostic Medical Microbiology - Traditional and Modern approachChhaya Sawant
Updated version of Diagnostic Microbiology - Traditional and Modern approach. The presentation is an overview of conventional techniques still used in many laboratories and new technologies such as Molecular- and Protein-based testing
Lab report that discusses the antigen-antibody precipitation reaction using the Ouchterlony Double Diffusion Technique.
Created by: Annisa Hayatunnufus
Bachelor of Pharmacy
Management & Science University
Immunoprecipitation: Procedure, Analysis and Applicationsajithnandanam
Immunoprecipitation is a precipitaion technique which allows the isolation of protein or protein complex from biological samples.
Incubate sample with antibody against protein of interest.
Separate antibody-protein complex from remaining sample
Analysis
Biol 390 – Lab 8 Restriction Digest and Gel Electrophoresis .docxmoirarandell
Biol 390 – Lab 8 Restriction Digest and Gel Electrophoresis
2
Objective
· Digest DNA of pGLO plasmid using restriction endonuclease enzymes.
· Run an agarose gel to separate the DNA fragments.
Background
Restriction enzymes cut DNA at specific sites generating a number of different sized fragments. The size of the fragments will depend on the number of sites the plasmid has and the specific enzyme used. The number of fragments can be predicted by viewing the map of the plasmid
Gel electrophoresis is a means of separating DNA in an electrical field. DNA is negatively charged and so will move to the anode (+). Larger fragments will move slower through the agarose matrix than the smaller molecules. Agarose is a polysaccharide polymer derived from seaweed: it is a purified from agar by removing the agaropectin component. Fragments are visualized using ethidium bromide, which will glow orange when exposed to UV light.
Materials
Restriction digest
· Restriction enzymes: Nhe1 and EcoR1 (New England Biolabs) – (KEEP ON ICE)
· Plasmid prepared in lab 7
· NanoDrop Lite spectrophotometer
· Microfuge tubes – Sterile
· 37 C degree bath – block heater
· Sterile 10ul and 200ul tips
· Bleach bottles for cleaning bench
· 10X NE Cut Smart Buffer – comes with enzyme
· Nitrile gloves
· Sterile DI water
· Shaved ice
· Ice block for enzymes
Gel Electrophoresis
· Agarose
· Sterile miliQ Water
· 15 well comb
· 50x TAE buffer
· DNA ladder – diluted in sample buffer (1 KB)
· Gel loading dye
· Gel electrophoresis chamber
· Power supply
· Ethidium bromide
· Gel Sys – visualization system
_______________________________________________
Procedure
Restriction Digest of plasmid DNA
· Safety: Wear nitrile gloves – prevent DNAase from your hands affecting the reaction and protect yourself from ethidium bromide
· Clean the bench with bleach - prevents exogenous enzymes interfering you’re your digests.
· Use the NanoDrop to determine the amount of DNA in your plasmid prep. Use this information to calculate how much sample you need to pipette into the reaction mix.
· Label an Eppendorf tube ‘+’ and another ‘-‘
· Make up a reaction mix in both tubes as follows for one of your plasmid samples
· add 1ug of DNA from your plasmid prep
· 5ul of 10X NE Cut Smart Buffer
· Sterile DI water to make the reaction mix to 50ul
For the + tube
· DNA
x ul
· 10X NE Cut Smart Buffer
5ul
· Nhe1 (add last to + tube)
1ul
· EcoR1 (add last to + tube)
1ul
· Sterile DI water
To make final volume to 50ul
· Add the restriction enzymes last to the + tube ONLY
· Repeat with the other two plasmid samples
For the – tube
· DNA
x ul
· 10X NE Buffer
5ul
· Nhe1
None
· EcoR1
None
· Sterile DI water
To make final volume to 50ul
· Do not add any enzyme to the ‘-‘ tube
· Repeat with the other two plasmid samples
· Mix the tubes by flicking – DO NOT VORTEX
· Give a 5 second spin in the centrifuge to bring the contents to the bottom
· Incu.
Escozine for Pets™ has 4 major production steps.
1. Collection of Scorpions from the Scorpion Reservation. 2. Extraction of venom, purification and therapeutic dose preparation. 3. Polarization of extract and quality control of Polarization 4. Manufacturing, quality control, warehouse and shipment.
For HPLC, sample solvents that adequately dissolve target compounds are required. Therefore, sample solvents that contain a high concentration of organic solvent are often used for reversed phase chromatography. The problem is that these solvents sometimes cause peak broadening.
This presentation discusses techniques for reducing the effects of sample solvents on UHPLC analyses.
ABSTRACT: The ELISA technique is a simple, sensitive, rapid, reliable, and versatile assay system for the quantitation of antigens and antibodies. Because of the extreme discriminating power of antibodies to recognize an almost infinite array of antigenic structures, the application of ELISA to analyte measurement is almost unlimited. ELISAs have been developed in many configurations depending on the particular application of the assay.
In solid-phase ELISA, one of the immunoreactants (antibody or antigen) is immobilized onto a solid support (microtiter plate) by adsorption, through non-covalent interactions. The immobilized antibody is then incubated with test solution containing the analyte of interest. Following a period of incubation and washing, the bound antigen is detected, by the addition of an enzyme-conjugated antibody that binds to the remaining antigenic sites on the antigen.
Although the technique is easy to perform and quite sensitive, there are certain problems to be solved before it becomes widely usable. In the present Memorandum the technical details are given and the advantages and shortcomings of the procedure are discussed. Present applications and future prospects are reviewed.
Symptoms like intermittent starting and key recognition errors signal potential problems with your Mercedes’ EIS. Use diagnostic steps like error code checks and spare key tests. Professional diagnosis and solutions like EIS replacement ensure safe driving. Consult a qualified technician for accurate diagnosis and repair.
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Here’s a handy guide to dashboard symbols so that you’ll never be confused again!
Save them for later and save the trouble!
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𝘼𝙣𝙩𝙞𝙦𝙪𝙚 𝙋𝙡𝙖𝙨𝙩𝙞𝙘 𝙏𝙧𝙖𝙙𝙚𝙧𝙨 𝙞𝙨 𝙫𝙚𝙧𝙮 𝙛𝙖𝙢𝙤𝙪𝙨 𝙛𝙤𝙧 𝙢𝙖𝙣𝙪𝙛𝙖𝙘𝙩𝙪𝙧𝙞𝙣𝙜 𝙩𝙝𝙚𝙞𝙧 𝙥𝙧𝙤𝙙𝙪𝙘𝙩𝙨. 𝙒𝙚 𝙝𝙖𝙫𝙚 𝙖𝙡𝙡 𝙩𝙝𝙚 𝙥𝙡𝙖𝙨𝙩𝙞𝙘 𝙜𝙧𝙖𝙣𝙪𝙡𝙚𝙨 𝙪𝙨𝙚𝙙 𝙞𝙣 𝙖𝙪𝙩𝙤𝙢𝙤𝙩𝙞𝙫𝙚 𝙖𝙣𝙙 𝙖𝙪𝙩𝙤 𝙥𝙖𝙧𝙩𝙨 𝙖𝙣𝙙 𝙖𝙡𝙡 𝙩𝙝𝙚 𝙛𝙖𝙢𝙤𝙪𝙨 𝙘𝙤𝙢𝙥𝙖𝙣𝙞𝙚𝙨 𝙗𝙪𝙮 𝙩𝙝𝙚 𝙜𝙧𝙖𝙣𝙪𝙡𝙚𝙨 𝙛𝙧𝙤𝙢 𝙪𝙨.
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11. Optimal mutual proportions (OMP) aka equivalent
proportions: usage of all available Ag and Ab in
formation of lattice
At OMP, addition of more Ag or Ab shows no
additional precipitation. All reactants participated
in initial reaction
12. Methodology:
Ab reacts with Ag for 1-2 h at 37*C to allow
rapid binding of Ab-Ag; forms small, soluble
complexes
13. Incubate for 24-28 h at 4*C to allow slow
development of small, soluble complexes from
step 1 into large insoluble complexes
14. Hence want to determine OMP= optimal
concentration of Ab and Ag to allow precipitin
reaction to occur
SO dilute Ag, keep Ab constant, measure amount
of precipitation, and determine dilution of Ag
that gives greatest amount of precipitation
Dilution of Ag that gives greatest precipitation =
OMP aka equivalence
15. • Change Ag amounts (mg), add to constant
amount of Ab, measure amount of
precipitation
• Plot amounts of precipitate VS amount of Ag
added
• To determine if a particular SN has excess Ag
or Ab, Add more Ag or Ab and see if additional
precipitation occurs
17. Agglutination Precipitin Reaction
Qualitative (slide test) Qualitative (ring test)
Ag – SRBC/Ab - α-SRBC Ag – OA /Ab – α-OA
Quantitative (microtiter Quantitative (microtiter
plate) assay)
Result: Titer Results: OMP
Serially diluted Ab Serially diluted Ag
More sensitive (need . Less sensitive (need
8-16mg) 80-320mg)
Ag is not soluble Ag is soluble
18. Serum: The clear liquid that can be separated
from clotted blood. Serum differs from
plasma, the liquid portion of normal unclotted
blood containing the red and white cells and
platelets. It is the clot that makes the
difference between serum and plasma.
Antiserum: antibody containing serum
19. Protocol
1) Aliquot 120µl of OA into a clean, dry
eppendorf tube and label accordingly. Set
aside for now.
120µl
OA
20. 2) Set up a 1:4 dilution (180µl of anti-OA serum +
540µl of PBS) in a clean, dry eppendorf tube and
label accordingly. Set aside for now.
540µl 180µl
PBS α-OA
22. 1:2
serial
of OA
bubble
dilution
(Minimize
1
formation and
change tips!!)
0.5
0.25
0.25
0.125
0.0625
0.03125
0.015625
0.0078125
0.00390625
0.00195312
5
0.00097656
25
23. When adding the serum solution, touch the
dispensing tip to the upper surface of the well
above the fluid level in the well.
50µl α-
OA
50µl
α-
OA
50µl
PBS
24. Immunodiffusion plate =
Immunodiffusion gel
Purpose: To test if the well showing
the largest amount of precipitate is
your OMP
Excess Ab
Your sample (centered on predicted
OMP)
25. Day 2
• Centrifuge plate (microtiter) 10 mins at 1200
rpm
• Prepare 2 plates (petri dishes): 10mL of agar to
each dish
• Label plates
• Incubate plates in fridge for 45-60 mins.
• Remove plates
26. Day 2
• Examine Day 1 plates
• Determine well(s) that show largest amount of
precipitate…OMP?
*Be very careful to not to disrupt the precipitate
(do not shake)!
32. Schedule a time to view your plate
tomorrow
If you know when you can make it, let me know
If not, find time tomorrow to come find me in
MH-317
I will go with you to view your
immunodiffusion
plate
33. Week 2 Day 1
Dilutions of Ag
Final Volume= 750µl
[OMP]
1 2 3 4 5
36. Week 2 Day 1
Excess OMP Excess
Ag Ab
1 2 3 4 5
37. Week 2 Day 2
Centrifuge at 1500 rpm, 4°C for 10 min.
Aspirate the SN_Resuspend pellet_Biorad
1 2 3 4 5
38. Calculations
•Construct a standard curve (total protein
concentration vs antigen concentration)
•Calculate the amount of antibody protein at
equivalence
• OMP, [ppt] = [antigen] + [antibody]
• Using the dilution of the original antiserum at
equivalence, calculate the amount of antibody
protein per ml of original undiluted antiserum
Editor's Notes
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http://classes.midlandstech.edu/carterp/Courses/bio225/chap18/Slide14.GIF. precipitate should form near interface between the two solutions= zone of optimal mutual proportions \n
excess Ab: all epitopes on Ag are covered by Ab- cock blocks subsequent lattice formation\nexcess Ag: has similar effect... so all Ab already bound to Ag and doesn't really care if new Ag is added-not sufficient Ab to cross--link Ag to form lattice, more precipitation does not occur\n
3) Deliver 50μl of PBS to wells 2 through 12 in row A\n Add 50 mL of the antigen (OA) solution (what you made in step 1) to wells 1 and 2 of row A. \n\n \n
\n
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If you have excess Ab in your sample, it will diffuse (migrate) toward the Ag you plated. If you have excess Ag it will diffuse (migrate) toward the Ab you plated. This will allow you to confirm or deny your predicted OMP. By plating the samples from surrounding wells of your microtitter plate, you will be able to identify the correct OMP.\n