Good news for milk chocolate admirers!

We all know the fact that dark chocolate is healthier than the milk one. But still most of us like to have a sweeter one and not the bitter one. Our craving lets us forget that milk chocolate lacks most of the natural and healthy antioxidants as they are lost during the making process. But soon we may not have to worry about the nutrition value of our favorite milk chocolate, all thanks to our researchers. Researchers at North Carolina State University in Raleigh found that peanut skins can be used to give the milk chocolate the same nutritional benefits as the dark chocolate. Moreover, this secret ingredient won't have any affect on the mind blowing taste.

Best things happen by chance.

Researchers didn’t start their study with the aim of finding a secret ingredient for the milk chocolate. In the United States, most peanut skins are frittered away while peanuts are used in making peanut butter. They were interested in finding a way to make good use of the wasted peanut skins.

They planned to extract antioxidants from the peanut skins. An antioxidant is a substance that inhibits the oxidation of other molecules. Studies have shown that antioxidant rich foods have a low risk of heart disease. However, antioxidants may impart a perceptible bitter taste to foods. Therefore, to disguise the bitter taste, they decided to mix the peanut skin extracts with the maltodextrin. Maltodextrin is a white powder made from corn, rice, potato starch or wheat and is used as a food additive.

Post the preparation of the antioxidant mixture, researchers added it to the milk chocolate. This addition brought the antioxidant levels in the milk chocolate equal to that of a dark chocolate without having hampered the taste of a candy.

This finding doesn’t indicate that one should turn chocolate into a staple food. Chocolate does contain fat and sugar and should not be consumed in large quantities. 

Reference:
L.L. Dean ​et​ ​al., 2016.​ ​Minimizing the negative flavor attributes and evaluating consumer acceptance of chocolate fortified with peanut skin extracts.​ ​​Journal of Food Science.​ ​​​81, ​S2824-S2830. 

Proteases

By Sarika Garg

Proteases are divided into five different groups, depending upon the type of molecule in the groove that carries out the actual work of catalysis. Serine proteases attack the peptide bond of the substrate using the hydroxyl group of the side chain of serine present in the catalytic centre. Threonine proteases act in the similar way. Cysteine proteases use the sulphur-hydrogen bond of a cysteine residue to initiate cleavage of the peptide bond. The acidic carboxyl groups of two aspartyl residues carry out this function in aspartyl proteases. Finally metalloproteases have a tightly bound zinc atom in their catalytic centre. Each of these groups of proteins is specialized to perform certain functions. For example, the coagulation of blood is the responsibility of serine proteases, whereas threonine proteases are essential, in particular, for the functioning of the proteasome, a large barrel-shaped protein-degrading apparatus in the cell. Programmed cell death is brought about by cysteine proteases. Digestion can not occur without aspartyl proteases and a subgroup of metalloproteases is essential for the breakdown and rebuilding of the extracellular matrix.

Proteases do not attack their protein substrates at random. Rather, they display a high degree of specificity. This specificity is not defined by the nature of the catalytic centre alone. On either side of the catalytic centre, there is a series of binding sites that favour particular amino acid in the substrate.    

Fig.: Specificity of protease binding. The specificity of protease binding is not defined solely by the nature of the catalytic centre. On either side of the catalytic centre there is a series of binding sites (S) that favour particular amino acids (P). This level of specificity ensures that proteases carry out their task only when truly needed.

Primary information retrieved from public and private sequencing projects, combined with data from the Merops, InterPro, and Ensembl databases, total of 553 genes have been annotated that encode proteases or protease homologues in the human genome. Mouse genome contains 628 proteases and protease homologues.

                                               Table: The human and mouse protease genes and pseudogenes.

References:

Lander, E.S., Linton, L.M., Birren, B., Nusbaum, C., et al., 2001. Initial sequencing and analysis of the human genome. Nature. 409(6822), 860-921.

Pietzsch, J., 2003. Signalling scissors: New perspectives on proteases. Horizon Symposia.

Puente, X.S., Sánchez, L.M., Overall, C.M., López-Otín, C., 2003. Human and mouse proteases: a comparative genomic approach. Nat Rev Genet. 4(7), 544-558.

Venter, J.C., Adams, M.D., Myers, E.W., Li, P.W., et al., 2001. The sequence of the human genome. Science. 291(5507), 1304-1351. 

Copyright © 2017 HS Counseling. All rights reserved. 

Shape your future by subsidising brain

Majority of the population invest in house, car, share market etc., believing that the investment they are doing is for the betterment of their future. But little do they know that these investments are fluctuating and are only the superficial ones.  It is like an exterior bubble which can be burst by mere crash of the property rates and stock market.  The most profitable investment one can ever make is investing in your brain.

Talking about the money, investment, future and brain, I have recollected one paraphrase, “Don’t run behind success and money, try to achieve excellence by sharpening your brain, knowledge and skills, success and money will automatically come looking for you”.

Truer words have never been spoken.

Brain is a very important organ in the human body as it allows a person to think, visualize, feel and store memories. Thinking and creative visualization are the key to success. Being more proficient will make you imperative in every area of your life. Inflation or recession, irrespective of the economy scenario, nurtured mind will always yield valuable returns.  The assured way to achieve a quality and successful life is to invest wisely in brain.

Learning new things, reading a lot, communicating with people from different backgrounds, participating in group discussions and playing educational games are some of the ways to foster your mental ability. These simple ways might help in brain development and function but might not be good enough to excel the brain activity. Here nootropics may be able to fill the gap. 

Nootropics are the drugs that help improve the mental functions.  These drugs hold the promise of helping with memory, cognitive ability, concentration, decision making, thinking and problem solving. These supplements are considered to work by amending availability of the brain’s supply of neurotransmitters, hormones and enzymes with escalated oxygen. Various nootropic drugs are available in the form of supplements. Apex GPA is a natural supplement and is even certified by Health Canada. If consumed as per the recommendation, it significantly improves memory, alertness and intellectual performances.

Investing in your mind truly shapes your future. It allows you to be skillful, proficient and compelling person.

Source: https://www.apexgpa.com/shape-future-subsidising-brain/

Smart drugs to revitalize intellectual power

In today’s competitive world, it won’t be wrong to say that ‘life is a race and everybody is contesting to win’. Three quintessential elements have been prominently reported to help gain an edge over the others which are:

-Having a good memory

-Being insightful

-Thinking on the feet

They sound too simple but aren’t naturally so commonplace. You must’ve found yourself in situations where you have two papers to write in a day and have completed its preparation 15 days in advance thinking that a short revision would do the trick. However, just the evening before…uh-oh… difficult to recall what you studied…unable to think new…lack of vision… Isn’t it?

How about getting smarter just by tossing a pill? It sounds too good to be true like in a TV commercial where you apply a muscle relief spray and get back on the soccer field in another 15 minutes. But ‘Smart drugs’ do exist and top notch researchers, globally, have proved the improvement in mental abilities on the ingestion of such drugs. Cognitive enhancers and Nootropics are the other names for Smart drugs.  

Smart drugs are the drugs that work on the human brain to improve memory, learning, attention, perception, reasoning, organization, social skills and vision. These drugs are often linked with neurological or mental disorders, but research evidence shows that regular people can get smarter by swallowing these drugs. Brain function and cognition is preserved by these drugs and hence, they serve as neuroprotective agents.

Various smart drugs are available in the form of supplements. Mechanism of action differs from one drug to the other and is very significant to consider. Age, dosage, duration, frequency and lifestyle are some of the key factors that should be taken into consideration before resorting to any drug. Any kind of negligence may alter the drug from being benignant to malignant.

Apex GPA supplements are composed of natural ingredients and are even certified by Health Canada. Scientific literature has reported that ingredients viz. Caffeine, Panax Ginseng and Ginkgo Biloba significantly improve calmness, alertness, concentration, attention, memory, accuracy and physical performance. If ingested as per the recommendation, you will never lag behind in terms of memory, concentration and alertness.

The underlying message is that the Smart drugs are innocuously beneficial if used with care.  

Source: https://www.apexgpa.com/smart-drugs-to-revitalize-intellectual-power/

Antibody purification

Buffers and Reagents:

Column: HiTrap NHS-activated HP 1 ml

Coupling buffer - 0.2 M NaHCO₃ , 0.5 M NaCl, pH 8.3

Buffer A - 0.5 M Ethanolamine, 0.5 M NaCl, pH 8.3

Buffer B - 0.1 M Sodiumacetate, 0.5 M NaCl, pH 4.0

1 mM HCl

2 M glycine-HCl pH 2.0

0.1% PBS/NaN₃

PBS

50 mM glycine-HCl pH 2.75

50 mM TAE pH 11.5

Buffer A - 0.5 M Ethanolamine, 0.5 M NaCl, pH 8.3

Buffer B - 0.1 M Sodiumacetate, 0.5 M NaCl, pH 4.0

Protocol:

Preparation of the peptide: Weigh 5 mg of the peptide and dissolve in 1 ml coupling buffer by vortexing.

Determine the protein concentration by measuring absorption at 280 nm using spectrophotometer.

Coupling to the column-

Fill 5 ml syringe with 1 mM HCl. Couple the column by placing the syringe on the yoke of the column.

Activation of the column-

Activate the column and wash away the isopropanol from the column by passing 6 ml of 1 mM HCl through the column. 

Coupling of the peptide-

Connect the column with two syringes, one on the upper side and other on the lower side. Fill the upper syringe with peptide solution and press it slowly on the column. The upper syringe will be empty and lower will be filled up half. Incubate the column for 1 hour at 4 ^oC. Then press the lower syringe slowly in order to move the solution to the upper syringe. Again incubate the set up at 4 ^oC for 1 hour. Move the peptide solution once every hour between two syringes for up to 4 hours. Incubate the whole set up at 4 ^oC overnight. 

Next day-

Remove the lower syringe and let the peptide solution drop out of the column. Preserve the flow through.

Washing of the column-

Wash the column twice using 1 ml coupling buffer. Preserve the flow through and mix it with the previous one. Mix 1 ml of the flow through with 1 ml of 2 M glycine-HCl pH 2.0 and measure the absorption at 280 nm. 

Determination of the coupling efficiency-

Volume of the coupled peptide solution:

A = A_{280, coupling solution} X V_{loaded volume of coupling solution}

Volume of the not coupled peptide:

B = A_{280, post-coupling wash after acidification} X V_{Volume post-column wash} X 2_{(dilution when acidified)}

Coupling yield, %:

(A-B)/A x 100 = Z

Z% of the peptide solution is coupled to the column. 

Denaturation of the column-

Denature the column by passing 2 ml of buffer A, 2 ml of buffer B and again 2 ml of buffer A. Repeat the sequence thrice and incubate the column at 25 ^oC for 30 minutes. Post 30 minutes, pass 2 ml of buffer B, 2 ml of buffer A and 2 ml of buffer B through the column and repeat the sequence thrice followed by incubation at 25 ^oC for 30 minutes.

Equilibration of the column-

Equilibrate the column by washing it with 10 ml of PBS once. 

Storage of the column-

Add 2 ml of 0.1% PBS/NaN₃ to the column and store the column at 4 ^oC.

Antibody purification-

1. Defrost the bleed and take 10 ml out. Freeze back the rest of the bleed at -80 ^oC.

2. Centrifuge the serum at 40,000 rpm, for 1 hour at 4 ^oC using ultracentrifuge.

L1 = clear, thick layer

L2 = pink, thin layer

3. Remove the L2 layer carefully with pasteur pipette and place it on ice.

4. Wash the coupled column with 10 ml of PBS.

5. Absorb the serum in a 5 ml syringe and place it on the column. Arrange the second syringe at the other end of the column.

6. Move the serum within the syringes every 15 minutes for up to 3 hours at 4 ^oC.

7. Collect the sample from the flow through.

8. Wash the column using 10 ml of PBS.

9. Elute the antibody with 10 ml of 50 mM glycine-HCl pH 2.75 and place the eluate on ice.

10. Again wash the column using 10 ml of PBS.

11. Elute the antibody with 10 ml of 50 mM TAE pH 11.5 and place the eluate on ice.

12. Wash the column using 20 ml of PBS and store the column in PBS/NaN₃ at 4 ^oC.

13. Concentrate the eluates to a volume of 4 ml using 30 kDa Amicon’s membranes. Thereafter, dialyse the eluates in PBS for 2 hours and then in PBS/50% Glycerol overnight at 4 ^oC. 

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