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.
Protein Immunoblotting- An Introduction to Western BlottingFatemeh Barantalab
Western blotting is an important technique used in cell and molecular biology. The specificity of the antibody-antigen interaction enables a target protein to be identified from a complex protein mixture. Western blotting can produce qualitative and semi-quantitative data about that protein. This powerpoint will attempt to explain the technique and theory behind western blot.
Potential problems of protein extraction using ripa buffer - Invent Biotechno...Invent Biotechnologies
Have your protein samples ready in a minute without altered protein profile and endogenous baseline that are usually associated with RIPA buffer. The Patented Spin Column Based Minute Protein Extraction Kits are fast, easy, and reliable. Sign Up for News and More Product Information.
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.
Protein Immunoblotting- An Introduction to Western BlottingFatemeh Barantalab
Western blotting is an important technique used in cell and molecular biology. The specificity of the antibody-antigen interaction enables a target protein to be identified from a complex protein mixture. Western blotting can produce qualitative and semi-quantitative data about that protein. This powerpoint will attempt to explain the technique and theory behind western blot.
Potential problems of protein extraction using ripa buffer - Invent Biotechno...Invent Biotechnologies
Have your protein samples ready in a minute without altered protein profile and endogenous baseline that are usually associated with RIPA buffer. The Patented Spin Column Based Minute Protein Extraction Kits are fast, easy, and reliable. Sign Up for News and More Product Information.
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.
Protein processing and production is often hampered by the formation of aggregates that restrict and complicate
the handling of proteins, antibodies and enzymes. NVoy is designed to minimise the sequential losses in consecutive
protein processing steps which would otherwise dramatically reduce the overall protein yield.
Measuring apoptosis in real time with a new luminescent methodMourad FERHAT, PhD
We developed a homogeneous luminogenic annexin V binding assay to detect apoptosis in real time using a multimode plate reader. The detection reagent has two different annexin V fusion proteins engineered to contain complementing domains of a binary luciferase, a substrate for luciferase and a cell impermeable fluorogenic DNA dye to detect necrotic cells. The method allow real-time monitoring of Cellular apoptosis and necrosis in microwell plates without washing steps with a highly sensitive luminescent signal. The AnnexinV luminescent method is amenable to High throughput and is a good alternative to FACS, low-throughput Annexin V-FITC based method.
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.
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.
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 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.
Protein processing and production is often hampered by the formation of aggregates that restrict and complicate
the handling of proteins, antibodies and enzymes. NVoy is designed to minimise the sequential losses in consecutive
protein processing steps which would otherwise dramatically reduce the overall protein yield.
Measuring apoptosis in real time with a new luminescent methodMourad FERHAT, PhD
We developed a homogeneous luminogenic annexin V binding assay to detect apoptosis in real time using a multimode plate reader. The detection reagent has two different annexin V fusion proteins engineered to contain complementing domains of a binary luciferase, a substrate for luciferase and a cell impermeable fluorogenic DNA dye to detect necrotic cells. The method allow real-time monitoring of Cellular apoptosis and necrosis in microwell plates without washing steps with a highly sensitive luminescent signal. The AnnexinV luminescent method is amenable to High throughput and is a good alternative to FACS, low-throughput Annexin V-FITC based method.
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.
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.
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 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.
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.
Western blotting WB (immunoblotting) is a widely practiced analytical technique to detect target proteins within samples using antigen-specific antibodies.
Western blotting WB (immunoblotting) is a widely practiced analytical technique to detect target proteins within samples using antigen-specific antibodies.
Flu Vaccine Alert in Bangalore Karnatakaaddon Scans
As flu season approaches, health officials in Bangalore, Karnataka, are urging residents to get their flu vaccinations. The seasonal flu, while common, can lead to severe health complications, particularly for vulnerable populations such as young children, the elderly, and those with underlying health conditions.
Dr. Vidisha Kumari, a leading epidemiologist in Bangalore, emphasizes the importance of getting vaccinated. "The flu vaccine is our best defense against the influenza virus. It not only protects individuals but also helps prevent the spread of the virus in our communities," he says.
This year, the flu season is expected to coincide with a potential increase in other respiratory illnesses. The Karnataka Health Department has launched an awareness campaign highlighting the significance of flu vaccinations. They have set up multiple vaccination centers across Bangalore, making it convenient for residents to receive their shots.
To encourage widespread vaccination, the government is also collaborating with local schools, workplaces, and community centers to facilitate vaccination drives. Special attention is being given to ensuring that the vaccine is accessible to all, including marginalized communities who may have limited access to healthcare.
Residents are reminded that the flu vaccine is safe and effective. Common side effects are mild and may include soreness at the injection site, mild fever, or muscle aches. These side effects are generally short-lived and far less severe than the flu itself.
Healthcare providers are also stressing the importance of continuing COVID-19 precautions. Wearing masks, practicing good hand hygiene, and maintaining social distancing are still crucial, especially in crowded places.
Protect yourself and your loved ones by getting vaccinated. Together, we can help keep Bangalore healthy and safe this flu season. For more information on vaccination centers and schedules, residents can visit the Karnataka Health Department’s official website or follow their social media pages.
Stay informed, stay safe, and get your flu shot today!
The prostate is an exocrine gland of the male mammalian reproductive system
It is a walnut-sized gland that forms part of the male reproductive system and is located in front of the rectum and just below the urinary bladder
Function is to store and secrete a clear, slightly alkaline fluid that constitutes 10-30% of the volume of the seminal fluid that along with the spermatozoa, constitutes semen
A healthy human prostate measures (4cm-vertical, by 3cm-horizontal, 2cm ant-post ).
It surrounds the urethra just below the urinary bladder. It has anterior, median, posterior and two lateral lobes
It’s work is regulated by androgens which are responsible for male sex characteristics
Generalised disease of the prostate due to hormonal derangement which leads to non malignant enlargement of the gland (increase in the number of epithelial cells and stromal tissue)to cause compression of the urethra leading to symptoms (LUTS
Title: Sense of Smell
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the primary categories of smells and the concept of odor blindness.
Explain the structure and location of the olfactory membrane and mucosa, including the types and roles of cells involved in olfaction.
Describe the pathway and mechanisms of olfactory signal transmission from the olfactory receptors to the brain.
Illustrate the biochemical cascade triggered by odorant binding to olfactory receptors, including the role of G-proteins and second messengers in generating an action potential.
Identify different types of olfactory disorders such as anosmia, hyposmia, hyperosmia, and dysosmia, including their potential causes.
Key Topics:
Olfactory Genes:
3% of the human genome accounts for olfactory genes.
400 genes for odorant receptors.
Olfactory Membrane:
Located in the superior part of the nasal cavity.
Medially: Folds downward along the superior septum.
Laterally: Folds over the superior turbinate and upper surface of the middle turbinate.
Total surface area: 5-10 square centimeters.
Olfactory Mucosa:
Olfactory Cells: Bipolar nerve cells derived from the CNS (100 million), with 4-25 olfactory cilia per cell.
Sustentacular Cells: Produce mucus and maintain ionic and molecular environment.
Basal Cells: Replace worn-out olfactory cells with an average lifespan of 1-2 months.
Bowman’s Gland: Secretes mucus.
Stimulation of Olfactory Cells:
Odorant dissolves in mucus and attaches to receptors on olfactory cilia.
Involves a cascade effect through G-proteins and second messengers, leading to depolarization and action potential generation in the olfactory nerve.
Quality of a Good Odorant:
Small (3-20 Carbon atoms), volatile, water-soluble, and lipid-soluble.
Facilitated by odorant-binding proteins in mucus.
Membrane Potential and Action Potential:
Resting membrane potential: -55mV.
Action potential frequency in the olfactory nerve increases with odorant strength.
Adaptation Towards the Sense of Smell:
Rapid adaptation within the first second, with further slow adaptation.
Psychological adaptation greater than receptor adaptation, involving feedback inhibition from the central nervous system.
Primary Sensations of Smell:
Camphoraceous, Musky, Floral, Pepperminty, Ethereal, Pungent, Putrid.
Odor Detection Threshold:
Examples: Hydrogen sulfide (0.0005 ppm), Methyl-mercaptan (0.002 ppm).
Some toxic substances are odorless at lethal concentrations.
Characteristics of Smell:
Odor blindness for single substances due to lack of appropriate receptor protein.
Behavioral and emotional influences of smell.
Transmission of Olfactory Signals:
From olfactory cells to glomeruli in the olfactory bulb, involving lateral inhibition.
Primitive, less old, and new olfactory systems with different path
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Ve...kevinkariuki227
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
Ozempic: Preoperative Management of Patients on GLP-1 Receptor Agonists Saeid Safari
Preoperative Management of Patients on GLP-1 Receptor Agonists like Ozempic and Semiglutide
ASA GUIDELINE
NYSORA Guideline
2 Case Reports of Gastric Ultrasound
micro teaching on communication m.sc nursing.pdfAnurag Sharma
Microteaching is a unique model of practice teaching. It is a viable instrument for the. desired change in the teaching behavior or the behavior potential which, in specified types of real. classroom situations, tends to facilitate the achievement of specified types of objectives.
Ethanol (CH3CH2OH), or beverage alcohol, is a two-carbon alcohol
that is rapidly distributed in the body and brain. Ethanol alters many
neurochemical systems and has rewarding and addictive properties. It
is the oldest recreational drug and likely contributes to more morbidity,
mortality, and public health costs than all illicit drugs combined. The
5th edition of the Diagnostic and Statistical Manual of Mental Disorders
(DSM-5) integrates alcohol abuse and alcohol dependence into a single
disorder called alcohol use disorder (AUD), with mild, moderate,
and severe subclassifications (American Psychiatric Association, 2013).
In the DSM-5, all types of substance abuse and dependence have been
combined into a single substance use disorder (SUD) on a continuum
from mild to severe. A diagnosis of AUD requires that at least two of
the 11 DSM-5 behaviors be present within a 12-month period (mild
AUD: 2–3 criteria; moderate AUD: 4–5 criteria; severe AUD: 6–11 criteria).
The four main behavioral effects of AUD are impaired control over
drinking, negative social consequences, risky use, and altered physiological
effects (tolerance, withdrawal). This chapter presents an overview
of the prevalence and harmful consequences of AUD in the U.S.,
the systemic nature of the disease, neurocircuitry and stages of AUD,
comorbidities, fetal alcohol spectrum disorders, genetic risk factors, and
pharmacotherapies for AUD.
Couples presenting to the infertility clinic- Do they really have infertility...Sujoy Dasgupta
Dr Sujoy Dasgupta presented the study on "Couples presenting to the infertility clinic- Do they really have infertility? – The unexplored stories of non-consummation" in the 13th Congress of the Asia Pacific Initiative on Reproduction (ASPIRE 2024) at Manila on 24 May, 2024.
These lecture slides, by Dr Sidra Arshad, offer a quick overview of physiological basis of a normal electrocardiogram.
Learning objectives:
1. Define an electrocardiogram (ECG) and electrocardiography
2. Describe how dipoles generated by the heart produce the waveforms of the ECG
3. Describe the components of a normal electrocardiogram of a typical bipolar leads (limb II)
4. Differentiate between intervals and segments
5. Enlist some common indications for obtaining an ECG
Study Resources:
1. Chapter 11, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 9, Human Physiology - From Cells to Systems, Lauralee Sherwood, 9th edition
3. Chapter 29, Ganong’s Review of Medical Physiology, 26th edition
4. Electrocardiogram, StatPearls - https://www.ncbi.nlm.nih.gov/books/NBK549803/
5. ECG in Medical Practice by ABM Abdullah, 4th edition
6. ECG Basics, http://www.nataliescasebook.com/tag/e-c-g-basics
Lung Cancer: Artificial Intelligence, Synergetics, Complex System Analysis, S...Oleg Kshivets
RESULTS: Overall life span (LS) was 2252.1±1742.5 days and cumulative 5-year survival (5YS) reached 73.2%, 10 years – 64.8%, 20 years – 42.5%. 513 LCP lived more than 5 years (LS=3124.6±1525.6 days), 148 LCP – more than 10 years (LS=5054.4±1504.1 days).199 LCP died because of LC (LS=562.7±374.5 days). 5YS of LCP after bi/lobectomies was significantly superior in comparison with LCP after pneumonectomies (78.1% vs.63.7%, P=0.00001 by log-rank test). AT significantly improved 5YS (66.3% vs. 34.8%) (P=0.00000 by log-rank test) only for LCP with N1-2. Cox modeling displayed that 5YS of LCP significantly depended on: phase transition (PT) early-invasive LC in terms of synergetics, PT N0—N12, cell ratio factors (ratio between cancer cells- CC and blood cells subpopulations), G1-3, histology, glucose, AT, blood cell circuit, prothrombin index, heparin tolerance, recalcification time (P=0.000-0.038). Neural networks, genetic algorithm selection and bootstrap simulation revealed relationships between 5YS and PT early-invasive LC (rank=1), PT N0—N12 (rank=2), thrombocytes/CC (3), erythrocytes/CC (4), eosinophils/CC (5), healthy cells/CC (6), lymphocytes/CC (7), segmented neutrophils/CC (8), stick neutrophils/CC (9), monocytes/CC (10); leucocytes/CC (11). Correct prediction of 5YS was 100% by neural networks computing (area under ROC curve=1.0; error=0.0).
CONCLUSIONS: 5YS of LCP after radical procedures significantly depended on: 1) PT early-invasive cancer; 2) PT N0--N12; 3) cell ratio factors; 4) blood cell circuit; 5) biochemical factors; 6) hemostasis system; 7) AT; 8) LC characteristics; 9) LC cell dynamics; 10) surgery type: lobectomy/pneumonectomy; 11) anthropometric data. Optimal diagnosis and treatment strategies for LC are: 1) screening and early detection of LC; 2) availability of experienced thoracic surgeons because of complexity of radical procedures; 3) aggressive en block surgery and adequate lymph node dissection for completeness; 4) precise prediction; 5) adjuvant chemoimmunoradiotherapy for LCP with unfavorable prognosis.
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Day2 western blot
1. Western blot sample preparation
Presented By
Dr. Hadeel khalaf Alboaklah
Dr. Muntader Mohammed
2. Western blot sample preparation
Sample preparation protocol contents
Lysis buffers
Protease and phosphatase inhibitors
Preparation of lysate from cell culture
Preparation of lysate from tissues
Determination of protein concentration
Preparation of samples for loading into gels: denatured and native, reduced and
non-reduced
3. Lysis buffers
Lysis buffers differ in their ability to solubilize proteins, with those containing
sodium dodecyl sulfate (SDS) and other ionic detergents considered to be the
harshest and therefore most likely to give the highest yield.
The main consideration when choosing a lysis buffer is whether the chosen
antibody will recognize denatured samples. When this is not the case, it will be
noted on the antibody datasheet, and buffers without detergent or with relatively
mild non-ionic detergents (NP-40, Triton X-100) should be used.
4. Protein location and lysis buffer choice
Protein location Buffer recommended
Whole cell NP-40
Cytoplasmic (soluble) Tris-HCl
Cytoplasmic (cytoskeletal bound) Tris-Triton
Membrane bound NP-40 or RIPA
Nuclear
RIPA or use nuclear fraction
protocol*
Mitochondria
RIPA or use mitochondrial fraction
protocol*
5. Lysis buffer recipes
NP-40 buffer
150 mM sodium chloride
1.0% NP-40 (Triton X-100 can be substituted for NP-40)
50 mM Tris pH 8.0
This is a popular buffer for studying proteins that are cytoplasmic or membrane-
bound, or for whole cell extracts. If there is concern that the protein of interest is not
being completely extracted from insoluble material or aggregates, RIPA buffer may be
more suitable as it contains ionic detergents that will more readily bring the proteins
into solution.
6. Protease and phosphatase inhibitors
As soon as lysis occurs, proteolysis, dephosphorylation and denaturation begin.
These events can be slowed down significantly if samples are kept on ice or at 4°C
at all times and appropriate inhibitors are added fresh to the lysis buffer.
Ready-to-use cocktails of inhibitors from various suppliers are available but you
can make your own cocktail.
7. Protease and phosphatase inhibitors
Inhibitor
Protease/
phosphatase
inhibited
Final concentration in
lysis buffer
Stock (store at -20°C)
Aprotinin
Trypsin, chymotrypsin,
plasmin
2 µg/mL
Dilute in water, 10
mg/mL. Do not re-use
thawed aliquots.
Leupeptin Lysosomal 5–10 µg/mL
Dilute in water. Do not
re-use thawed
aliquots.
Pepstatin A Aspartic proteases 1 µg/mL
Dilute in methanol, 1
mM.
8. Preparation of lysate from cell culture
Place the cell culture dish on ice and wash the cells with ice-cold PBS.
Aspirate the PBS, then add ice-cold lysis buffer (1 mL per 107 cells/100 mm
dish/150 cm2 flask; 0.5 mL per 5x106 cells/60 mm dish/75 cm2 flask).
Scrape adherent cells off the dish using a cold plastic cell scraper, then gently
transfer the cell suspension into a pre-cooled microcentrifuge tube. Alternatively
cells can be trypsinized and washed with PBS prior to resuspension in lysis buffer
in a microcentrifuge tube.
9. Preparation of lysate from cell culture
Maintain constant agitation for 30 min at 4°C.
Centrifuge in a microcentrifuge at 4°C. You may have to vary the centrifugation
force and time depending on the cell type; a guideline is 20 min at 12,000 rpm but
this must be determined for your experiment (e.g. leukocytes need a very light
centrifugation).
Gently remove the tubes from the centrifuge and place on ice, aspirate the
supernatant and place in a fresh tube kept on ice, and discard the pellet.
10. Preparation of lysate from tissues
Dissect the tissue of interest with clean tools, on ice preferably, and as quickly as possible to
prevent degradation by proteases.
Place the tissue in round-bottom microcentrifuge tubes or Eppendorf tubes and immerse in liquid
nitrogen to snap freeze. Store samples at -80°C for later use or keep on ice for immediate
homogenization.
For a ~5 mg piece of tissue, add ~300 μL of ice cold lysis buffer rapidly to the tube, homogenize
with an electric homogenizer, rinse the blade twice with another 2 x 300 μL lysis buffer, then
maintain constant agitation for 2 h at 4°C (eg place on an orbital shaker in the fridge). Volumes of
lysis buffer must be determined in relation to the amount of tissue present. Protein extract should
not be too diluted to avoid loss of protein and large volumes of samples to be loaded onto gels.
The minimum recommended concentration is 0.1 mg/mL, optimal concentration is 1–5 mg/mL).
Centrifuge for 20 min at 12,000 rpm at 4°C in a microcentrifuge. Gently remove the tubes from the
centrifuge and place on ice, aspirate the supernatant and place in a fresh tube kept on ice. Discard
the pellet.
11. Determination of protein concentration
Perform a Bradford assay, a Lowry assay or a bicinchoninic acid (BCA) assay.
Bovine serum albumin (BSA) is a frequently used protein standard.
Once you have determined the concentration of each sample, you can freeze
them at -20°C or -80°C for later use or prepare for immunoprecipitation or for
loading onto a gel.
12. Preparation of samples for loading into
gels
Denatured, reduced samples:
Antibodies typically recognize a small portion of the protein of interest (referred to as
the epitope) and this domain may reside within the 3D conformation of the protein.
To enable access of the antibody to this portion it is necessary to unfold the protein,
ie denature it.
To denature, use a loading buffer with the anionic detergent sodium dodecyl sulfate
(SDS), and boil the mixture at 95–100°C for 5 min. Heating at 70°C for 5–10 min is
also acceptable and may be preferable when studying multi-pass membrane proteins.
These tend to aggregate when boiled and the aggregates may not enter the gel
efficiently.
13. To denature
The standard loading buffer is called 2X Laemmli buffer (Laemmli UK, 1970. Cleavage of structural
proteins during the assembly of the head of bateriophage T4. Nature, 227, 680–5). It can also be
made at 4X and 6X strength to minimize dilution of the samples. The 2X is to be mixed in 1:1 ratio
with the sample.
2x Laemmli buffer recipe
4% SDS
10% 2-mercaptoethanol
20% glycerol
0.004% bromophenol blue
0.125 M Tris HCl
Check the pH and bring it to pH 6.8
14. Why use these substances
When SDS is used with proteins, all of the proteins become negatively charged by
their attachment to the SDS anions. SDS binds to proteins fairly specifically in a
mass ratio of 1.4:1. In doing so, SDS confers a negative charge to the polypeptide
in proportion to its length. Denatured polypeptides become rods of negative
charge with equal charge densities per unit length. Therefore, migration is
determined by molecular weight, rather than by the intrinsic charge of the
polypeptide.
SDS grade is important for high-quality protein separation: a protein stained
background along individual gel tracts with indistinct or slightly distinct protein
bands are indicative of old or poor quality SDS. Inclusion of 2-mercaptoethanol or
dithiothreitol in the buffer reduces disulphide bridges, which is necessary for
separation by size.
15. Why use these substances
Glycerol is added to the loading buffer to increase the density of the sample to be
loaded and hence maintain the sample at the bottom of the well, restricting overflow
and uneven gel loading.
To visualize the migration of proteins it is common to include a small anionic dye
molecule in the loading buffer (eg bromophenol blue). Since the dye is anionic and
small, it will migrate the fastest of any component in the mixture to be separated and
provide a migration front to monitor the separation progress.
During protein sample treatment the sample should be mixed by vortexing before and
after the heating step for best resolution.
