This document provides information on protein isolation and quantification methods. It discusses extracting proteins from plant, animal, fungal and bacterial tissues using physical and chemical lysis methods. The steps include tissue collection, grinding, centrifugation, and precipitation. It also describes separating proteins by SDS-PAGE and quantifying concentration spectrophotometrically. The goal of protein extraction is to recover proteins for analysis and study their structure, function and interactions.
Yeast two-hybrid is based on the reconstitution of a functional transcription factor (TF) when two proteins or polypeptides of interest interact. Upon interaction between the bait and the prey, the DBD and AD are brought in close proximity and a functional TF is reconstituted upstream of the reporter gene.
wo-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of gel electrophoresis commonly used to analyze proteins. Mixtures of proteins are separated by two properties in two dimensions on 2D gels. 2-DE was first independently introduced by O'Farrell and Klose in 1975.
Yeast two-hybrid is based on the reconstitution of a functional transcription factor (TF) when two proteins or polypeptides of interest interact. Upon interaction between the bait and the prey, the DBD and AD are brought in close proximity and a functional TF is reconstituted upstream of the reporter gene.
wo-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of gel electrophoresis commonly used to analyze proteins. Mixtures of proteins are separated by two properties in two dimensions on 2D gels. 2-DE was first independently introduced by O'Farrell and Klose in 1975.
STS stands for sequence tagged site which is short DNA sequence, generally between 100 and 500 bp in length, that is easily recognizable and occurs only once in the chromosome or genome being studied.
A presentation covering the process of protoplast culture including protoplast isolation, protoplast fusion, culture of protoplast, its application, factors affecting protoplast culture and the future of protoplasts.
PAGE is a subtype of the gel electrophoresis whereby the normal gel is replaced with polyacrylamide gels use as the support matrix.
widely used and has very much importance.
COMPLETE PROCEDURE & USES are described in the slide.
What is Genome,Genome mapping,types of Genome mapping,linkage or genetic mapping,Physical mapping,Somatic cell hybridization
Radiation hybridization ,Fish( =fluorescence in - situ hybridization),Types of probes for FISH,applications,Molecular markers,Rflp(= Restriction fragment length polymorphism),RFLPs may have the following Applications;Advantages of rflp,disAdvantages of rflp, Rapd(=Random amplification of polymorphic DNA),Process of rapd, Difference between rflp &rapd
Open reading frame is part of reading frame that contains no stop codons or region of amino acids coding triple codons.
ORF starts with start codon and ends at stop codon.
It is a subtype of the gel electrophoresis whereby the normal gel is replaced with polyacrylamide gels used as support media.
Gels are made by free radical-induced polymerization of acrylamide and N,N’-Methylenebisacrylamide.
It is the most widely used technique of electrophoresis.
Overcoming Key Challenges of Protein Mass Spectrometry Sample PreparationMourad FERHAT, PhD
Overcoming Key Challenges of Protein Mass Spectrometry Sample Preparation
Bottom-up proteomics is widely accepted as a primary method to characterize proteins. To ensure efficient protein analysis researchers must optimize key steps in the workflow to avoid potential pitfalls such as poor protein sample preparation and inconsistent LC-MS instrument performance. In this presentation, we will:
• Investigate the cause of incomplete trypsin digestion and solution to this problem.
• Discuss the advantage of alternative proteases for mass spec protein analysis.
• Review the impact of mass spec compatible surfactants on protein digestion in gel and protein extraction from animal tissues.
• Detail new reference mass spec protein and peptide materials designed to optimize protein sample preparation steps and monitor key instrument performance parameters.
The presentation should prove valuable to any researcher using bottom-up proteomics, and who is concerned with improving protein mass spec sample preparation and mass spec instrument performance.
STS stands for sequence tagged site which is short DNA sequence, generally between 100 and 500 bp in length, that is easily recognizable and occurs only once in the chromosome or genome being studied.
A presentation covering the process of protoplast culture including protoplast isolation, protoplast fusion, culture of protoplast, its application, factors affecting protoplast culture and the future of protoplasts.
PAGE is a subtype of the gel electrophoresis whereby the normal gel is replaced with polyacrylamide gels use as the support matrix.
widely used and has very much importance.
COMPLETE PROCEDURE & USES are described in the slide.
What is Genome,Genome mapping,types of Genome mapping,linkage or genetic mapping,Physical mapping,Somatic cell hybridization
Radiation hybridization ,Fish( =fluorescence in - situ hybridization),Types of probes for FISH,applications,Molecular markers,Rflp(= Restriction fragment length polymorphism),RFLPs may have the following Applications;Advantages of rflp,disAdvantages of rflp, Rapd(=Random amplification of polymorphic DNA),Process of rapd, Difference between rflp &rapd
Open reading frame is part of reading frame that contains no stop codons or region of amino acids coding triple codons.
ORF starts with start codon and ends at stop codon.
It is a subtype of the gel electrophoresis whereby the normal gel is replaced with polyacrylamide gels used as support media.
Gels are made by free radical-induced polymerization of acrylamide and N,N’-Methylenebisacrylamide.
It is the most widely used technique of electrophoresis.
Overcoming Key Challenges of Protein Mass Spectrometry Sample PreparationMourad FERHAT, PhD
Overcoming Key Challenges of Protein Mass Spectrometry Sample Preparation
Bottom-up proteomics is widely accepted as a primary method to characterize proteins. To ensure efficient protein analysis researchers must optimize key steps in the workflow to avoid potential pitfalls such as poor protein sample preparation and inconsistent LC-MS instrument performance. In this presentation, we will:
• Investigate the cause of incomplete trypsin digestion and solution to this problem.
• Discuss the advantage of alternative proteases for mass spec protein analysis.
• Review the impact of mass spec compatible surfactants on protein digestion in gel and protein extraction from animal tissues.
• Detail new reference mass spec protein and peptide materials designed to optimize protein sample preparation steps and monitor key instrument performance parameters.
The presentation should prove valuable to any researcher using bottom-up proteomics, and who is concerned with improving protein mass spec sample preparation and mass spec instrument performance.
SeedEZ 3D culture methods and protocols - total protein extractionLena Biosciences
SeedEZ 3D cell culture methods and protocols – total protein extraction and quantification. 3D cell culturing conditions influence protein extraction from cells for downstream analysis. Depending on extraction buffer used, an amount of protein from the extracellular matrix (in addition to cell protein) may be extracted. For these reasons, setup suitable controls. Here, it is shown how to extract total protein from cells cultured in 3D in the SeedEZ under different culturing conditions and how to apply a protein assay to quantify it. Protein assay results with 3D brain co-culture models comprising primary cortical neurons and primary-harvested and one-time passaged mixed glia (astrocytes and microglia) seeded into (a) uncoated SeedEZ substrate, (b) Poly-D-Lysine coated SeedEZ substrate, and (c) seeded in 7.5 mg/ml protein suspension into uncoated SeedEZ are shown.
The first step in sample preparation is isolating proteins from their source. Usually, proteins are isolated from cells or tissues via lysis. Lysis breaks down the cell membrane to separate proteins from the non-soluble parts of the cell. A number of lysis buffers can be used to prepare samples for western blotting.
The extraction of DNA involves three main steps that are cell lysis, protein separation, and DNA purification. Cell lysis is usually performed by incubation of cell in buffer containing detergent and protease. Cellular proteins are salted out or phase separated using organic solvents. Finally DNA is isolated and purified either by alcohol precipitation or adsorption with silica and elution.
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.
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.
Similar to Protein isolation and quantification (17)
Cotton, known as “White Gold”, is the premier commercial crop in India. Among the different constraints that limit the yield of cotton in India, insect pests are considered to be the most serious. Among these insect pests nowadays, Whitefly, Bemisia tabaci (Gennadius) is most important. It is highly polyphagous pest and feeds on over 600 plant species including many agricultural crops (Oliveira et al., 2001). During last week of September, 1994 the whitefly assumed an epidemic form on cotton and brinjal crops at farmers fields throughout the Haryana state (Sharma and Batra, 1995). There are 24 different biotypes of whitefly. It transmits more than 111 species of plant pathogenic viruses (Jones, 2003). There are many approaches for controlling this pest viz., physical, cultural,biotechnological, biological, chemical, biopesticides and biorationals. Yellow sticky traps in various forms can catch large no. of whiteflies (Gerling and Horowitz, 1984). Use of light emitting diodes increase the attractiveness, specificity and adaptability of these visual traps (Stukenberg, 2014). There are cultural practices such as avoidance in time, avoidance in space and behavioural manipulations to manage whiteflies (Hilje et al., 2001). A reflective mulch (also called silver and metallic) treatment resulted in a lower incidence of adult whiteflies as compared with a standard black mulch treatment (Simmons et al., 2010). Biopesticides such as fungi and azadirachtin are also used to manage whitefly. In pot culture, 2% concentration of mineral oil + neem oil and mineral oil + Pongamia glabra seed oil were effective against Bemisia tabaci with a mean population reduction of 81.83% and 81.52% respectively (Chandra Shekhar et al., 2015). Five species of predators : Serangium parcesetosum, Brumoides suturalis, Cheilomenes sexmaculata, Coccinella septempunctata, Chrysoperla zastrowi and a parasitoid, Encarsia lutea were identified in Haryana (Kedar et al., 2014). Pyriproxyfen 10 EC @ 125gm a.i/ha was found most effective Insect Growth Regulator against whitefly (Kumar et al., 2014). Imidacloprid proved to be the most effective insecticide against whitefly upto seven days after application (Afzal et al., 2014). Spiromesifen 240 SC @ 0.4 ml/lt followed by buprofezin 10 EC @ 1.0 ml/lt were found as the most effective treatments with more than 75 per cent mean reduction in nymphal population of whiteflies (Maha Lakshmi et al., 2015). A chitin inhibitor gene Tma12 from a fern Tectaria spp. was identified for whitefly defence. RNA interference (RNAi)- mediated gene silencing was explored for the control of Bemisia tabaci (Upadhyay et al., 2011).
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3. Protein extraction
Protein
Proteins are the biomolecules,
composed of amino acid, forming the
building block of the system and performs
most of the biological functions of the system.
Protein extraction:
The process by which the proteins from the cell are recovered for
the analysis purpose is called protein extraction
4. Why ?
Proteins are extracted from tissues for a wide range of reasons, including
To compare the structure of proteins as expressed by different
organism.
To purify a protein in order to identify the gene that encodes it, and
To resolve proteins by SDS-PAGE.
To assay an enzyme in a crude extract for physiological studies.
To study the mechanism of action of an enzyme.
To diagnose parasitic diseases.
5. Generic outline for protein isolation
1. Collection of sample, washing, cleaning and grinding to fine powder
2. Cell lysis with physical (Grinding) and chemical (lysis buffers)
methods.
3. Separation of proteins from non-protein components (nucleic acids
and lipids) & recovery of bulk proteins from a crude extract
4. Further separation of target protein-containing fraction from the bulk
proteins
5. Recovery of the target protein in a highly purified state along with a
high yield
6. Materials
Frozen or fresh plant material
Liquid nitrogen
Extraction buffer
Mortar & pestle or tissue grinder
20- to 70-μm pore size nylon mesh
Refrigerated centrifuge and centrifuge tubes
Extraction of Proteins from Plant Tissues
7. procedure
Cleaning and weighing of Leaves
Take isolated leaves in a petri-plate & wash with distilled water to
remove all the particulate matter on the surface.
Dry the leaves on a tissue paper without applying excess pressure.
After the leaves are totally dry, weigh the required amount on a
balance.
8. Liquid Nitrogen Treatment
Take a clean mortar and pestle and place it on ice.
Pre-chill the mortar with liquid nitrogen.
Transfer the leaves into the pre-chilled mortar & Add around 10
ml of liquid nitrogen, all at one go.
9. Grinding the Leaves & Lysis Buffer Treatment
Grind the leaves till a fine powder is seen.
To the powdered leaves, add lysis buffer and grind thoroughly.
Transfer the lysate to fresh eppendorf tubes & Add some more lysis
buffer, to ensure proper lysis of cells.
Vortex the tube thoroughly to mix the contents uniformly.
Protein Precipitation at -20°C
After vortexing the contents, incubate the tube at -20°C for 1
hour.
The proteins at the end of this incubation, get precipitated in the
buffered solution.
10. Centrifugation and Acetone Wash
Take the tube out of -20oC freezer. Centrifuge the tube at 14000
rpm, 4oC for 30 minutes,
Take the tube out of the centrifuge. The tube can be seen to have
a clearly demarcated pellet and supernatant. Discard the
supernatant carefully without disturbing the pellet.
11. To the pellet, add 1ml of wash buffer. Wash buffer helps in
removing the color of the pellet.
Vortex the tube to thoroughly let the pellet dissolve into the buffer.
Incubate the tube in the -20o freezer for 30 minutes.
Remove the tube from the freezer carefully. Centrifuge the tube at
14000rpm, 4oC for 15 minutes.
Discard the supernatant carefully
without disturbing the pellet. Air dry
the pellet to remove even traces of
acetone.
12. Rehydration Buffer Treatment
Add 400 μL of rehydration buffer to the air-dried pellet.
Vortex the tube till the pellet dissolves in the rehydration buffer
(CHAPS solubilizes the proteins, Urea denatures the proteins.)
Incubate the tube overnight at 4oC for complete solubilisation of
proteins in the rehydration buffer.
Sample Storage
The sample contains the protein
If not required immediately it can be stored at -20°C for later use.
13. Protein isolation from animal tissues
Materials
Waring blender
Refrigerated centrifuge
Polypropylene screw-cap
centrifuge tubes
14. Procedure
1. Trim fat and connective tissue from the body and cut into small
pieces
2. Place the tissue in the precooled blender vessel and add cold
extraction buffer(2)
3. Homogenize at full speed for 1—3 min depending on the
toughness of the tissue.
4. Pour the homogenate into a glass beaker, place on ice, and stir
for 15-30 min to ensure full extraction.
15. 5. Remove cell debis and other particulate matter from the
homogenate by centrifugation at 4°C.
6. Pour off the supernatant carefully to avoid disturbing sedimented
material. Any fatty material that has floated to the top of the tube
should be removed by filtering the extract through cheese cloth
7. The pellet may be re-extracted with more buffer to increase the
yield.
8. The filtrate obtained at this stage may require further treatment
to remove insoluble material
9. Thus obtained is protein extract.
16. Protein extraction from fungi
Materials
Malt extract agar (MEA)
Glucose yeast medium (GYM)
Pectin broth
Tris-glycine buffer
Pectinase gel
Gel buffer
Electrode buffer
0.1 Malic acid
17. procedure
This protocol describes the extraction of aqueous intracellular proteins
from the filamentous fungus Metarhizium anisopliae
Harvest the mycelium from given culture by vacuum-assisted filtration onto
Whatman No. 3 filter paper.
Wash the mycelium once in the Buchner funnel with sterile deionized water,
and transfer the harvested mycelium from the filter paper to a plastic Petri dish
with a sterilized spatula.
Freeze the mycelium at -20°C for 24 h.
18. Disrupt freeze dried mycelium by briefly grinding it in a mortar
and pestle & collect it in sterile 1.5 mL micro-centrifuge tubes
(~500-mg). Rehydrate it in 1 mL of Tris-glycine buffer.
Clarify the slurry by centrifugation at 12,500 g for 40 min at 4°C.
After centrifugation, collect the supernatant into another sterile
micro-centrifuge tube
The collected supernatant will contain the total cytoplasmic
proteins. The samples as prepared here typically contain 15-100
mg/mL protein
19. Protein extraction form Bacteria
Materials
Lysis buffer
Hen egg lysozyme
DNase 1
Sodium deoxycholate
MgCl2
20. procedure
1. Harvest the bacterial cells by centrifugation at 1000g for 15
min at 4°C, and pour off the supernatant.
2. Weigh the wet pellet.
3. Add approx 3 mL of lysis buffer for each wet gram of bacterial
cell pellet and resuspend.
4. Add lysozyme to a concentration of 300 µg/mL and stir the
suspension for 30 min at 4°C
21. 5. Add deoxycholate to a concentration of 1 mg/mL while stirring.
6. Place at room temperature, and add DNase 1 to a concentration of
10 mg/mL and MgCl2 to 10 mM. Stir suspension for a further 15
min to remove the viscous nucleic acid
7. Centrifuge the suspension at 10,000g for 15 min at 4°C.
8. Resuspend the pellet in lysis buffer to the same volume as the
supernatant, and analyze aliquots of both for the protein of
interest on SDS-PAGE
22. SDS-PAGE analysis
SDS-PAGE : sodium dodecyl sulfate
polyacrylamide gel electrophoresis
It is a common technique used to separate,
visualize, and therefore compare the
relative amount of individual polypeptide
chains contained in different fractions
It yields the mass of each individual
subunit derived from the denatured
complex
23. procedure
Denaturation:
A small sample from each fraction is first mixed with an excess of
SDS, ßME and bromophenol blue.
Each mixture is then placed in a boiling water bath for several
minutes
In the end, each polypeptide chain is unfolded into a negatively-
charged, rod-shaped complex with a relatively constant charge
to mass ratio.
24. Electrophoresis:
Take a solid, rectangular gel of
polyacrylamide that is cross-
linked to a desired mesh or size
exclusion limit.
A small aliquot from each
denatured sample is transferred
to separate wells of gel.
An electric field is applied across the loaded gel with the positive pole
positioned on the opposite side of the samples
25. Negatively-charged complexes get drived the through the mesh of
polyacrylamide which effectively filters them according to the length of
their Stokes radius as they wiggle through the porous matrix to the
opposite side of the gel.
Small particles move through
the gel more rapidly than larger
ones,
The electrophoresis is visually
followed. Power supply is shut off before any protein in the sample reaches
the bottom of the gel.
26. Staining and destaining:
Following electrophoresis, the entire gel is soaked in a stain
solution containing a dye that tightly binds to the backbone of
each denatured polypeptide chain (usually Coomassie brilliant
blue).
Excess stain is then washed from the gel by soaking it in a destain
solution long enough for the blue-stained bands of each
polypeptide to be visualized against the clear, colorless
background of the gel.
27. PAGE of a sample proteins
stained with Coomassie
blue
Two SDS-PAGE-gels after a completed run
28. After staining, different species
biomolecules appear as distinct bands
within the gel.
It is common to run molecular weight
size markers of known molecular
weight in a separate lane in the gel to
calibrate the gel
Approximate molecular mass of
unknown biomolecules is determined
by comparing the distance traveled
relative to the marker.
Picture of an SDS-PAGE. The
molecular markers (ladder) are
in the left lane
29. protein concentration by spectrophotometer
This method is recommended for pure protein solutions
Principle
Proteins in solution absorb UV light with absorbance maxima at
280 and 200 nm. Amino acids with aromatic rings are the
primary reason for the absorbance peak at 280 nm. Peptide
bonds are primarily responsible for the peak at 200 nm.
30. Procedure
1. Warm up the UV lamp (about 15 min.)
2. Adjust wavelength to 280 nm
3. Calibrate to zero absorbance with
buffer solution only
4. Measure absorbance of the protein solution
5. Adjust wavelength to 260 nm
6. Calibrate to zero absorbance with buffer
solution only
7. Measure absorbance of the protein solution
31. Analysis
Unknown proteins or protein mixtures.
Concentration(mg/ml) =
Absorbance at 280 nm
path length (cm.)
Pure protein of known absorbance coefficient: for 1cm path length
concentration =
Absorbance at 280 nm
absorbance coefficient
32. Concentration may in mg/ml, %, or molarity depending on type
coefficient is used.
Mg protein/ml =
% protein
10
=
molarity
Protein Molecular weight
Unknowns protein with possible nucleic acid contamination.
Conc. (mg/ml) = (1.55 x A280) - 0.76 x A260)