Estimation of protein using Bicinchoninic acid (BCA)

This method was developed by Smith et al in 1985 and thereafter, Pierce Chemical Company exploited it by developing a kit. In this method, there is reduction of cupric ions to cuprous ions by the protein under alkaline condition. The cuprous ions are detected by using bicinchoninic acid (BCA). The macromoecular structure of the protein, the number of peptide bonds and the presence of four amino acids (cysteine, cystine, tyrosine, tryptophan) are responsible for color development in protein samples. Accordingly, protein concentrations generally are determined and reported with reference to standards of a common protein such as bovine serum albumin (BSA).

Reagents: 

BCA Protein Assay kit is available from Pierce, Rockford, Il 61105, USA. The reagent kit has the following reagents:

Reagent A (sodium carbonate, sodium bicarbonate, bicinchoninic acid and sodium tartarate in 0.1 M sodium hydroxide)

Reagent B (4% cupric sulfate)

Albumin Standard Ampules, 2 mg/ml: Prepare bovine serum albumin (BSA) at 2.0 mg/ ml in 0.9% saline and 0.05% sodium azide

Just before use, prepare working BCA reagent by mixing one part of Reagent B with 50 parts of Reagent A. 

Protocol:

Spectrophotometer-

Prepare a series of dilutions of known concentration from the protein and assay alongside the protein sample of unknown concentration.

1. Prepare standard series by taking aliquots of the BSA protein in different tubes so that amount of the protein in different tubes varies from 5 to 50 μg protein.

2. Take aliquot of protein and water to make the volume 1.0 ml.

3. Prepare the blank by taking 1.0 ml of water.  

4. Add 2 ml of working BCA reagent in each tube and shake well.

5. Incubate all the tubes at 37^oC for 30 minutes.

6. Measure the absorbance at 562 nm in a spectrophotometer.

7. Taking into account the readings with standard proteins, plot a calibration curve taking absorbance on the vertical axis and amount of the protein on the horizontal axis.

8. From this plot, using absorbance value with the unknown protein, determine the amount of the protein in the unknown sample.

Plate reader-

1. Prepare standard series by taking aliquots of the BSA protein in different wells so that amount of the protein varies from 0-25 μg protein. 

Standard (Volume in µl)

H2O	25	20	15	10	0
BSA (1 µg/µl)	0	5	10	15	25

2. Take aliquot of protein and water to make the volume 25 µl.

3. Add 200 µl of working BCA reagent in each well and place the plate on shaker at 37 ^oC for 30 minutes in dark.

4. Measure the absorbance at 562 nm using plate reader.

5. Taking into account the readings with standard proteins, plot a calibration curve taking absorbance on the vertical axis and amount of the protein on the horizontal axis.

6. From this plot, using absorbance value with the unknown protein, determine the amount of the protein in the unknown sample.

Although the method has several advantages viz. compatibility with ionic and non-ionic detergents, stable working reagent, less protein to protein variation, broad linear working ranges and good sensitivity; reducing agents and copper chelators do interfere with this assay. This problem can be ruled out by removing the interfering substances prior to BCA analysis by precipitating the protein sample with TCA or acetone and re-dissolving the protein pellet in NaOH or water. Some people prefer to bind proteins to positively charged nylon at alkaline pH and washing out the non-bound interfering agents.

Hello

I have worked in a lab for around 10 years and I remember how I and my other fellow colleagues so wanted to have all the protocols at one place. I am sure this is what exactly all researchers wish to have. Isn't it?

In this internet era, if I want to find electrophoresis protocol, it is not difficult. Simply google the appropriate terms and I will get 100s of results in return. Similarly, for other protocols viz. immunoprecipitation, immunohistochemistry etc., I will just have to google the terms. But every time to find a protocol I have to make a separate research and choose the best hit from all the return results. Isn't it tedious? Also, as a researcher I so wish to have several lab protocols for the same technique at one place so that I can manipulate the protocol for the best results possible. Don't you think the same way?

I am sure you do. Therefore, I have started this blog where my efforts are to cumulate all the scientific protocols. However, I alone cannot achieve the aforementioned dream to have all scientific protocols and different protocols for the same technique at one place. Therefore, I hereby invite you all to join this social venture and post your protocols as well along with your name and affiliation. Your contribution is significant. Let's serve the scientific society and change the world for better.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

Perform SDS-PAGE for separation of proteins according to a modified protocol (Laemmli, 1970; Matsudaira and Burgess, 1978). Sodium dodecyl sulfate, an anionic detergent, is used to completely suppress the native charge on the proteins. It gives them a large negative coat of detergent molecules. The SDS also interacts with the hydrophobic core of proteins, it causes a rapid and irreversible unfolding that linearizes the polypeptide chains which all now have a roughly equivalent charge/mass ratio. Therefore, in SDS-PAGE, the separation of proteins is on the basis of molecular weight (size) of the proteins.

Buffers and Reagents:

Lower Gel Stock Solution (1.0 M Tris-HCl, pH 8.8; 0.25% SDS)

60.6 g Tris-HCl

1.25 g SDS

Dissolve in H₂O, adjust the pH 8.8 with HCl and make the volume 500 ml with H₂O.

Upper Gel Stock Solution (0.5 M Tris-HCl, pH 6.8; 0.25% SDS)

15.2 g Tris-HCl

0.63 g SDS

Dissolve in H₂O, adjust the pH 6.8 with HCl and make the volume 250 ml with H₂O.

1X SDS Running Buffer (0.19 M Glycine; 25 mM Tris-HCl, pH 8.3; 0.1 % SDS)

7.2 g Glycine

1.5 g Tris-HCl

0.5 g SDS

Dissolve in H₂O, adjust the pH 8.3 and make the volume 500 ml with H₂O.

5X SDS Sample Buffer (0.25 M Tris-HCl, pH 6.8; 50% Glycerol; 5% SDS and 0.1% Bromophenol blue)

1.2 g Tris-HCl

20 ml Glycerol

10 ml of 20% SDS

0.04 g Bromophenol blue

Dissolve in H₂O, adjust the pH 6.8 and make the volume 40 ml with H₂O.

** CAUTION ** SDS powder is hazardous. Prepare solution in a ventilated fume hood. 

Protocol:

For casting gels, a system with vertically oriented glass plates with 1 mm spacer in between can be used. Prepare separating gel (small pore size gel) and stacking gel (large pore size gel) as first described by Ornstein, 1964.

Composition of separating and stacking gel:

Components	Separating gel	Separating gel	Stacking gel (4%) (ml)
	10% (ml)	17% (ml)
40% Acrylamide/ Bis acrylamide (37.5:1)	15	25.6	5.4
Tris HCl (1.0 M, pH 8.8)	22	22	-
Tris HCl (0.25 M, pH 6.8)	-	-	27
10% SDS	0.6	0.6	0.54
TEMED	0.12	0.12	0.108
10% APS	0.065	0.065	0.065
H2O	22	11.5	20.9

First pour the separating gel between glass plates and apply a layer of isopropanol on it. After polymerization of the separating gel, remove the layer of isopropanol and wash the polymerized gel with H₂O. Then pour the stacking gel on the top of the polymerized separating gel and insert the comb. When the gel solution gets solidified, place the gel in the electrophoresis chamber filled with the 1X SDS running buffer and remove the comb. Mix the protein sample to be electrophoresed with 5X SDS sample buffer and denature it completely by heating at 95^oC for 5 minutes. Then load the denatured protein samples along with the molecular weight marker proteins and perform the electrophoresis at 150 volts. Stop the current when the bromophenol blue tracking dye reaches at the end.

Molecular weight protein markers that can be used:

Protein name	Molecular weight (kDa)
β-Galactosidase	116.0
Bovine serum albumin	66.2
Lactate-dehydrogenase	45.0
Restriction endonuclease Bsp981	35.0
Lactoglobulin	18.0
Lysozyme	14.4

Computer-brain implant for the first time!

Computer-brain implant for the first time!
58 year old woman paralyzed by Amyotrophic lateral sclerosis (ALS) has become the first patient to use a computer-brain interface. Netherlands doctors have performed the successful brain implant on the lady which has enabled her to communicate in a day-to-day life via a speech computer. 
The lady was completely locked-in due to the severe nerve degeneration and was only able to control her eye muscles. 
A device called as electrocorticograph that has electrodes fitted in the brain has been installed on the women's brain. Brain activity is detected by the electrodes and signals are transmitted. The lady has a screen in front of her where she can compose words by using her brain to click the mouse at the proper time. She can spell out the messages at two letters per minute. The words composed by a lady are then uttered by a speech computer. The entire transmission is wireless. 
This implant has opened doors of hope for many patients and their kiths and kins. 

Marijuana could help alcoholics and drug addicts to sweat it out!

Marijuana could help alcoholics and drug addicts to sweat it out!
Research study at University of British Columbia in collaboration with Florida State University, National Centre for PTSD and Centre for Innovation and Implementation in California found that the use of marijuana could help treat alcoholics, drug addiction and mental health. Medical Cannabis may also help with symptoms of depression, post-traumatic stress disorder and social anxiety. However, cannabis might not be suggested for conditions such as bipolar disorder and psychosis.
Marijauna legalization in Canada could be possible early next year and therefore it is crucial to understand the advantages and disadvantages of cannabis to help health professionals.

Belt-type medical device for diabetes and obesity

Abdomen belt for diabetes and obesity treatment!
Exercise is the most effective treatment for both diabetes and obesity but sometimes it gets difficult for the older generation or for the overweight population. Researchers at Kumamoto University have developed a medical device that can be simply attached to the abdomen and can effect visceral fat loss and improve blood glucose (sugar).
The heat shock response (HSR) function is decreased in the population with type 2 diabetes. Researchers found that by restoring the function of HSP72, the main protein of HSR, improved glucose-related abnormalities. They found that HSP72 can be activated by a combination of mild electrical stimulation (MES) with heat shock (HS).
The scientists developed a wearable medical device that can be used as an abdomen belt. This device used a special type of rubber to transmit MES and HS at the same time. On performing a clinical trial, decrease in fasting glucose levels, loss of visceral fat, improve insulin resistance, and a significant improvement in glycated hemoglobin values was observed.
This device can be expected to producxe large therapeutic effect.

Ticklish spot identified in the rat brain

Ticklish spot identified in the rat brain!
Why are we ticklish? Why can't we control our laughter in response to tickling? These are the most inexplicable ticklish doubts discussed by great academicians including Aristotle and Charles Darwin. Despite of all the efforts, the mechanism remained mysterious.
The new research conducted at Humboldt-Universität zu Berlin reported that rats enthusiastically emitted numerous calls in response to tickling. Belly and underneath feet are the most ticklish areas in rats. The responses rats emitted after tickling are the similar calls emitted during playing. Researchers observed that the nerve cells in the trunk region of the somatosensory cortex, a large brain structure that contains an ordered representation of the body and handles stimuli on the body, responded strongly to tickling. Similar brain responses were observed during play behaviors when rats were not even touched by the scientist. On making rats apprehensive, the activity in the nerve cells and the ticklishness were reduced. These results suggested that cells in the somatosensory cortex represents ticklish sensation.
In researcher's own words, 'Perhaps ticklishness is a trick of the brain that rewards interacting and playing'.

Sleep and dreaming genes identified

Sleep and dreaming genes have been recognized!
Researchers at University of Texas Southwestern Medical Center in Dallas and University of Tsukuba in Japan have identified two genes, Salt-Inducible Kinase 3 Sik3 (Sik3) and Sodium Leak Channel Non-selective (Nalcn), that control deep sleep and dreaming.
REM (Rapid eye movement) is a stage that makes up around 25% of a night’s sleep and dreaming is supposed to occur during REM sleep. Whereas NREM (Non-rapid eye movement) accounts for the remaining 75% of the sleep and it is a stage of deep sleep.
The genes identified by the researchers regulates the amount of REM and NREM sleep in mammals. Study involved more than 8000 mice and reported that sleepy mice had a mutation in Sik3 gene whereas dreamless mice had a mutation in Nalcn gene.
This discovery might be beneficial for new treatments for sleep disorders.

Spinach plants can detect explosives

Spinach is well known as an excellent source of Vitamin K, Vitamin A, manganese, folate, magnesium, iron, copper, vitamin B2, vitamin B6, vitamin E, calcium, potassium and vitamin C. It is a very good source of dietary fiber, phosphorus, vitamin B1, zinc, protein and choline.
But did you know that spinach plants can also detect explosives?
Engineers at MIT have embedded the spinach leaves with carbon nanotubes and have transformed the spinach plants into sensors that can detect explosives and wirelessly transmit that information to a small computer similar to a smartphone. Plants were designed to detect chemical compounds known as nitroaromatics, which are often used in landmines and other explosives. When one of these chemicals is present in the groundwater sampled naturally by the plant, carbon nanotubes embedded in the plant leaves emit a fluorescent signal that can be read with an infrared camera. The camera can be attached to a small computer similar to a smartphone, which then sends an email to the user.
This discovery has overpower the plant/human communication barrier.

Dengue vaccines may escalate zika outbreaks

Dengue vaccines may escalate zika outbreaks!
Researchers at York University and Xi’an Jiaotong University identified that vaccinations for dengue increase the number of people contracting zika. They have also shown that more people in a particular population that are vaccinated against dengue, the earlier and larger the Zika outbreak. The research also found that the most effective way to minimize the unintended effect of dengue vaccinations on Zika outbreaks is through an integrated strategy that includes mosquito control.
More work needs to be done to understand how to optimize dengue vaccination programs and minimize the risk of Zika outbreaks.

Sweet spot identified in the brain for the pain relief

Sweet spot in the brain for the pain relief!
Researchers at Northwestern University have identified the region in the brain responsible for the placebo effect in pain relief. This sweet spot of the pain killing placebo effect has been identified for the first time and could result in the design of more personalized medicine for patients with chronic pain.
Physicians treat patients’ pain by either changing dosage or trying alternative drug if one doesn’t work. This new technology will help physicians to see what part of the brain is activated during an individual’s pain and choose the specific drug to target this spot. They will also be able to measure how the patient’s pain region is affected by the drug.

Omega-3 fatty acids promote the clearance of brain metabolites

Fish oil promotes the clearance of metabolites from the brain!
New research published in The FASEB Journal reports that Omega-3 polyunsaturated fatty acids, found in fish oil, could improve the function of the glymphatic system, which advances the clearance of waste from the brain, and promote the clearance of metabolites including amyloid beta peptides, a primary culprit in Alzheimer’s disease.
Omega-3 polyunsaturated fatty acids help maintain the brain homeostasis, which may provide benefits in a number of neurological diseases, such as Alzheimer’s disease, traumatic brain injury, and sleep impairment, among others.

Alternative drug to chemotherapy for advanced lung cancer

Alternative drug to chemotherapy for advanced lung cancer!
Researchers at University of California, LA evaluated an immunotherapy drug, Pembrolizumab.
The drug has been approved by the U.S. Food & Drug Administration as first-line treatment for non-small cell lung cancer (NSCLC). The first line treatment means that the drug can be accessed by people without first having to receive treatments viz. chemotherapy.
This drug will allow patients to live longer while delaying, and in some cases potentially avoiding, the side effects of traditional chemotherapy.

Hypertension is linked to air pollution and traffic noise

Air pollution and traffic noise is linked to hypertension!
Researchers at European Society of Cardiology (ESC) investigated the effects of both air pollution and traffic noise by following over 41,000 people in five different countries for five to nine years.
A total of 41,072 people living in Norway, Sweden, Denmark, Germany and Spain participated in the study, which was part of the “European Study of Cohorts for Air Pollution Effects” (ESCAPE) project that is investigating long-term effects of exposure to air pollution on human health in Europe.
The study found that air pollution and noise is associated with an increase in cases of hypertension. Both the air pollution and noise affect different, or not completely overlapping, pathways involved in disturbances in the way the body normally functions.

Oxygen levels can reset the circadian clocks

Circadian clock- this term is recollected only when one is jetlagged.
But scientists are curious to study the driving mechanism of biological rhythm. Light, food, and temperature are the known indications to influence circadian rhythms. But recently oxygen has also joined the list.
A new study published in Cell Metabolism has reported that circadian clocks of mice can be modulated by variations in surrounding oxygen levels. Researchers have shown that changing the oxygen levels by just 3%, twice a day, will synchronize mouse cells to a circadian rhythm. They have also surmised a protein HIF1α as a link between oxygen and circadian clock. Cells with low HIF1α levels won’t synchronize in response to oxygen variations.

Role of glial cells in appetite control

Glial cells- a novel participant in regulating appetite.
Researchers at MIT have reported that brain cells called glial cells play a fundamental role in appetite control and feeding behavior. In mice, they found that glial cells activation triggers overeating and on suppression of the cells, hunger is also suppressed.
This finding could open a way for developing drugs against obesity and other associated disorders.

Alzheimer’s disease breakthrough

Breakthrough made by the researchers at the University of Minnesota!
Alzheimer’s disease most common sign is memory loss and with the new breakthrough Alzheimer’s disease itself might soon be a forgotten memory.
Specific enzyme, Caspase-2, has been discovered as the culprit in this disease. It attacks the brain neurons and targets a specific protein in the brain called Tau. Tau fragment generated by caspase-2 cleavage accumulates in neurons.
They found that by preventing the caspase-2 cleavage of Tau or by reducing the caspase-2 levels, memory deficits could be recovered in mice. This is a significant step forward in the fight against Alzheimer’s disease and memory loss.

Live animals from artificial eggs

Live animals created from artifical eggs grown in a petri dish!
Japanese Scientists breakthrough is remarkable. They took tissue cells from the tail of a mouse, reprogrammed them as stem cells and then turned them into eggs in the lab. Thereafter, the eggs were fertilized and the resulting embryos were implanted in female mice. This led to the birth of 11 healthy pups.
This might also be relevant for the human application in the future.

2016 World Food Prize

2016 World Food Prize has been awarded to the four scientists- Dr. Maria Andrade, Dr. Robert Mwanga, Dr. Jan Low and Dr. Howard Bouis.
They are recognised for their work developing the vitamin-enriched orange-fleshed sweet potato. They have enriched the sweet potatoes using biofortification process making it a valuable source of calories and nutrients for millions of people.
In the developing world, Vitamin A deficiency is the most harmful forms of malnutrition which is the leading cause of blindness, maternal mortality, weak immunity, etc. More than 140 million pre-school children and more than 7 million pregnant women in several nations gets affected by the Vitamin A deficiency.
Biofortification improves the nutritional quality of food crops through agronomic practices, conventional plant breeding, or modern biotechnology. It can be the way to reach out to the populations where supplementation and conventional fortification activities are difficult to implement.

2016 Nobel Prize in Literature

US singer Bob Dylan is the recipient of 2016 Nobel Prize in Literature.
The 75 year old megastar is recognized for his creation of new poetic expressions within the great American song tradition.
He is the first one to have won the prestigious award for writing songs.