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Friday, April 29, 2011

Principle of Protein Assays

Principle of Protein Assays - Fc -Lowry -Bradford assays

Principle of Protein Assay

Numerous spectrophotometric methods have been developed to estimate the amount of protein in a sample. Proteins are chromophores with absorption maximum in the UV range.Some proteins, such as cytochromes and hemoglobin, will have distinct spectral characteristics due to prosthetic groups. In addition, several indirect ways to measure protein concentrations
spectrophotometrically have been developed.

UV Absorption

 A simple method to measure protein concentration is to determine the absorption at 280nm. Tyrosine and tryptophan residues which have Amax at 275 and 280, respectively, are responsible for this absorption. The distribution  of tyrosine and tryptophan is fairly constant among proteins so it is not absolutely necessary to determine an extinction coefficient for each individual protein. Typically, a 1 mg/ml protein solution will result in an A280 of approximately one (1). In the case of purified proteins the exact extinction coefficient will depend on the exact amount of tyrosine and tryptophan in that particular protein. For example, a 1 mg/ml IgG solution has an A280 of approximately 1.5. The simplicity and ability to completely recover the sample are the major advantages of this method. The lower limit of sensitivity for UV absorption is 5-10 µg/ml. 

 A potential problem with using A280 values to calculate protein concentration is the absorption due to contaminating substances, and in particular, nucleic acids which have an Amax at 260 nm. It is possible to use correction factors that permit the determination of protein concentrations. A particularly convenient formula is:  

   (A235 - A280)/2.51 = mg/ml protein.

Indirect spectrophometric assays (eg., Lowry, Bradford) for the determination of protein concentrations overcome some of the problems associated with interfering substances in protein samples. However, the measured protein cannot  be recovered in such assays and they take longer to perform. 

Folin-Ciocalteu or Lowry

 Historically, one of the most widely used protein assays was the Lowry assay. This assay is a modification of a previous assay known as the Biuret. In the Lowry assay proteins react with alkaline Cu2+ reducing it to Cu+. The reduced Cu+ and the side-chain (R) groups of tryptophan, tyrosine and cysteine react with the Folin-Ciocalteu reagent (complex of inorganic salts) to form a blue color that is proportional to the amount of protein. The A600-750 is determined and protein concentration is calculated from a standard curve. The assay is linear 1-300  µg and the lower limit of sensitivity is 1-5  µg/ml. Substances in the sample such as detergents can interfere with the results and therefore appropriate controls and blanks need to be carried out.

Bradford or Coomassie Blue G-250

Protein assay

The Bradford assay has replaced the Lowry as the standard protein assay. The major advantage is that it is carried out in a single  step and that there are very few interfering substances. The principle of the assay is based on a shift of the Amax of the Coomassie-blue (G-250) dye from 465 nm to 595 nm in the presence of protein due to a stabilization of the anionic form of the dye. The dye reacts primarily with arginine residues and to lesser extent with his, lysine, tyrosine, tryptophan and phenylalanine.  Protein concentrations are determined by developing a standard curve with known amounts of proteins and extrapolating the absorbance
values of the samples. The standard curves are not linear over a wide range of protein concentrations. This assay can also carried out in 96-well plates and read on automated ELISA readers. Programs are available that will automatically calculate the protein concentrations
based upon a standard curve.

Assay Of Specific Proteins

In addition to measuring the total amount of protein, it is often necessary to estimate the amount of a specific protein in a mixture of proteins. Measuring a specific protein will depend upon the availability of an assay that is specific for the protein of interest. Protein assays should be practical in addition  to being specific and accurate . Typically protein assays are based upon the biological activity of the protein of interest. For example, enzyme assays will detect the conversion of a substrate to a product. Enzymes assays can be based upon colorimetric, fluorescent or radioactive substrates (or products). Many proteins bind to ligands or other substances and this binding activity is measured. Bioassays measure a change in some biological property (eg., stimulation of cell division). In cases where the protein of interest has no measurable activity or the activity is unknown it may be possible to generate antibodies against the protein and develop an immunoassay (to be discussed later). If antibodies against such a  protein are not available, the assay may simply be the amount of a protein band on a Commassie blue-stained gel following electrophoresis.


Lecture Notes for Methods in Cell Biology

Thursday, April 28, 2011

DNA Origami : Big Impact on Nanotechnology

While the primary job of DNA in cells is to carry genetic information from one generation to the next, some scientists also see the highly stable and programmable molecule as an ideal building material for nanoscale structures that could be used to deliver drugs, act as biosensors, perform artificial photosynthesis and more.

Via Science Daily

Wednesday, April 27, 2011

Preparation of Competent Cells for Transformation

  1. Inoculate 2ml of LB with a single DH5 colony. Incubate culture overnight at 37oC while shaking at 250 RPM.

  2. The following morning, inoculate 500ml of LB with 1ml of saturated overnight culture. Incubate culture at 37oC while shaking at 250RPM until OD600 = 0.5 (3-5 hours).

  3. Transfer culture to 2 pre-chilled sterile 250ml centrifuge tubes. Pellet bacteria cells with a 5000 RPM spin for 10 minutes at 4oC. Discard supernatant. Place pellets on ice.

  4. Resuspend cells in 10ml cold CaCl2 solution. Pool cells together into one pre-chilled 50ml Oakridge tube.

  5. Pellet cells with a 2500 RPM spin for 5 minutes at 4oC. Discard supernatant and resuspend cells in 10ml cold CaCl2 solution. Set on ice 30 minutes.

  6. Pellet cells with a 2500 RPM spin for 5 minutes at 4oC. Discard supernatant and resuspend cells in 2ml cold CaCl2 solution.

*At this point you can leave cells on ice overnight at 4oC – this increases competency in some cases*

  1. Dispense cells into 50ul aliquot in pre-chilled sterile polypropylene tubes. Store cells at –80oC.

Test for Competency


  1. Remove competent DH5a cells from the –80oC and immediately place on ice. Once thawed, add >10ng of plasmid DNA to a 50ul aliquot of competent cells. Place cells/DNA on ice for 3 minutes.

  2. Heat shock cells at 42oC for 3 minutes.

  3. Place cells back on ice for 3 minutes.

  4. Add 1ml LB to cells/DNA. Tape tube onto shaking incubator platform and incubate cells/DNA for 1 hour at 37oC while shaking at 250 RPM.

  5. Pellet cells with a quick spin. Remove 800ul of supernatant. Resuspend cells in the remaining supernatant.

  6. Plate 100ul and 200ul of transformation onto 2 LB+Amp plate. Place plates inverted at 37oC overnight.


Scientists Create Stable, Self-Renewing Neural Stem Cells

In a paper published in the April 25 early online edition of the Proceedings of the National Academy of Sciences, researchers at the University of California, San Diego School of Medicine, the Gladstone Institutes in San Francisco and colleagues report a game-changing advance in stem cell science: the creation of long-term, self-renewing, primitive neural precursor cells from human embryonic stem cells (hESCs) that can be directed to become many types of neuron without increased risk of tumor formation.

"It's a big step forward," said Kang Zhang, MD, PhD, professor of ophthalmology and human genetics at Shiley Eye Center and director of the Institute for Genomic Medicine, both at UC San Diego. "It means we can generate stable, renewable neural stem cells or downstream products quickly, in great quantities and in a clinical grade -- millions in less than a week -- that can be used for clinical trials and, eventually, for clinical treatments. Until now, that has not been possible."

Via Science Daily

Solar Power Goes Viral: Researchers Use Virus to Improve Solar-Cell Efficiency

Researchers at MIT have found a way to make significant improvements to the power-conversion efficiency of solar cells by enlisting the services of tiny viruses to perform detailed assembly work at the microscopic level.

The new MIT research, published online in the journal Nature Nanotechnology, is based on findings that carbon nanotubes -- microscopic, hollow cylinders of pure carbon -- can enhance the efficiency of electron collection from a solar cell's surface.

Graduate students Xiangnan Dang and Hyunjung Yi -- working with Angela Belcher, the W. M. Keck Professor of Energy, and several other researchers -- found that a genetically engineered version of a virus called M13, which normally infects bacteria, can be used to control the arrangement of the nanotubes on a surface, keeping the tubes separate so they can't short out the circuits, and keeping the tubes apart so they don't clump.

Via Science Daily

SDS PAGE : Protocol

SDS-PAGE - Protocol


SDS PAGE - Sodium dodecyl sulphate polyacrylamide gel electrophoresis, is an electrophoretic technique used to separate out proteins based on the molecular weigtht.

1. The electrophoresis apparatus was assembled by taking two glass plates (Bangalore Genei), spacers are kept in between the two plates, bottom is sealed with sealing gel.

Electrophoretic Unit  

2. The stock solution :
a) 0.5M Tris (pH 6.8)
- 6.05 g tris in 100ml distilled water; pH was adjusted with conc. HCl
b) 1.5 M Tris (pH 8.8)
- 18.15 g Tris in 100ml distilled water; pH was adjusted with conc. HCl
c) 10% SDS
- 1g in 10ml 
3. The working solution :
a) Solution A (30%)
Acrylamide – 29.2 g
Bisacrylamide – 0.8 g
b) 10% APS – 100mg in 1ml
c) electrophoresis buffer (1L)
SDS – 1g
Tris – 3g
Glycine – 14.4g
4. Sampling buffer :
I. reducing -> a) 0.5 Tris (pH 6.8) – 0.5ml
b) glycerol – 1ml
c) ß mercaptoethanol – 1ml
d) SDS – 0.2ml
e) Bromophenol Blue (BPB)– a pinch
II. non-reducing -> a) 0.5M Tris (pH 6.8) – 1ml
b) glycerol – 1.5ml
c) SDS – 0.2g
d) Bromophenol Blue (BPB)– a pinch
5. Separating gel (12%) :
a) distilled water – 2.8ml
b) Tris – 1.25ml
c) acrylamide – 0.83ml
d) SDS – 75 µl
e) APS – 52.5ml
f) TEMED – 6 µl
6. stacking and sealing gel (same composition for both) :
a) distilled water (D/W)– 2.8ml
b) Tris – 1.25ml
c) acrylamide – 0.83ml
d) SDS – 50 µl
e) APS – 50 µl
f) TEMED – 5 µl
7. The lower portion of the assembly sealed by using the sealing gel. The separating gel was poured in between the plates and allowed to solidify. After solidification the stacking gel was poured and placed the comb into the stacking gel for making wells. Allow it to solidify. After solidification the electrophoresis buffer was poured into the apparatus. The sample was added into the wells. 
Sample Preparation :
Mix 20ul of sample with 5ul of sample buffer
BSA : 1mg/ml - 10ul + 10ul DW + 5ul of sample buffer
Protein Mol.wt Marker - see the provider catalogue and dilute accordingly.
Vortex all the samples, boil @ 100 ºC for 15 mins. and centrifuge for 2-3mins.
Load the samples into wells and note down the order of loading.
8. Switch on the Electrophoretic power supply with 160v, 40mA electricity, run the gel till it touches the sealing gel. After separation of bands, switch off the current and remove the gel carefully for staining.

 CBB ( Coomassie Brilliant Blue ) staining : CBB R-250
a) CBB – 0.25g
b) methanol – 50ml
c) acetic acid – 10ml
d) D/W – 40ml
Dip the gel in CBB stain and leave it on a gel rock for a few mins.
CBB Destaining :
a) methanol – 50ml
b) acetic acid – 10ml
c) D/W – 40ml
Keep the gel in destaining solution in rocker, destain till the protein bands are clearly visible.


A CBB Stained Gel

Silver staining :
- wash the run gel with D/W.
- rock with 50ml of 50% methanol for 15 mins.(twice)
- rinse once with D/W for 10 seconds.
- wash with silver solution for 15 mins.
- wash 5 times with D/W (1 min. duration each)
Silver staining solution :
a) add 200mg of AgNO3 in 50ml of D/W.
b) add 1ml of 1M NaOH and mix the solution.
c) add 650 µl of liquid ammonia and add immediately to the gel. Shake for 15mins. 

Developing solution:
a) add 250 µl of 1% citric acid.
b) 50ml of water
c) 50 µl of formaldehyde. 


A Silver Stained Gel

Note: Some follow slightly different procedure in buffer making and sample preparation.