Gyurng-Soo Yoo

Studies of structure activity relationship of flavonoids for the anti-allergic actions

The structure activity relationship of flavonoids for anti-allergic actions was studied by determining the IC50 values for the degranulation. The hexosaminidase release from RBL-2H3 cells (degranulation marker) was employed as an estimate for the anti-allergic actions. Among 22 flavonoid compounds tested, luteolin, apigenin, diosmetin, fisetin, and quercetin were found to be most active with IC50 values less than 10 μM.

Direct Blue 71 staining of proteins bound to blotting membranes

A sensitive staining method for protein blots using Direct Blue 71 is described. It is based on the selective binding of dye molecules to proteins in acidic solution and produces bluish violet colored bands. It is a simple and rapid procedure, involving only staining and rinsing steps that occur within 7 min. The sensitivity of this method is 5—10 ng of protein on nitrocellulose (NC) and 10—20 ng on polyvinylidene difluoride (PVDF), which is tenfold better than that of the commonly used Ponceau S staining. Moreover, the staining is reversible for subsequent immunostaining, without impairing immunoreactivity. To remove the dye from the developed bands, changes in pH and hydrophobicity of the solvent are required. Due to its sensitivity, rapidity, simplicity, and low cost, this stain may be more practical than other dye‐based stains or metal‐based stains for routine laboratory purposes.

Counterion‐dye staining method for DNA in agarose gels using crystal violet and methyl orange

Sensitive and safe methods for visualization of DNA in agarose gels are described. 0.001% crystal violet dissolved in distilled water was used for DNA staining on agarose gels and it could detect as little as 16 ng of DNA (3 kb, pGem‐7Zf/EcoRI) without destaining procedure. The detection limit is four times lower than that of ethidium bromide. To improve the sensitivity, we studied a counterion‐dye staining method using methyl orange as a counterion‐dye which contributes to reduce excessive background staining by crystal violet. Dye concentration, pH of staining solution, mixing molar ratio of two dyes, and staining times were optimized for the counterion‐dye staining. By the staining with a mixed solution of 0.0025% crystal violet and 0.0005% methyl orange in distilled water, 8 ng of the 3 kb DNA in an agarose gel was detected within 30 min.

Fast protein staining in sodium dodecyl sulfate polyacrylamide gel using counter ion-dyes, coomassie brilliant blue R-250 and neutral red

A fast and sensitive protein staining method in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using both an acidic dye, Coomassie Brilliant Blue R-250 (CBBR) and a basic dye, Neutral Red (NR) is described. It is based on a counter ion-dye staining technique that employs oppositely charged two dyes to form an ion-pair complex. The selective binding of the free dye molecules to proteins in an acidic solution enhances the staining effect of CBBR on protein bands, and also reduces gel background. It is a rapid staining procedure, involving fixing and staining steps with short destaining that are completed in about 1 h. As the result, it showed two to fourfold increase in sensitivity comparing with CBBR staining. The stained protein bands can be visualized at the same time of staining.

Evans Blue Staining Method for Detection of Proteins on Polyacrylamide Gels with Rhodamine B

Abstract A new staining method employing a mixed dye technique is simple, rapid, and sensitive. Solutions of Evans blue (EB, 0.01 %) and rhodamine B (RB, 0.5 %) were mixed up with 1:1 volume ratio. This mixed dye staining method detects as little as 10 ng of bovine serum albumine (BSA). EB is used for protein staining based on its structural similarity to Coomassie brilliant blue R–250 (CB). RB acts as a supplementary agent which inhibits the binding of EB to gel matrix, and so increases the staining effect of EB and enables to achieve cutting down staining/destaining times.

An Eosin Y Method for Protein Determination in Solution

ABSTRACT A method for protein determination using eosin Y is described. Eosin Y reacts with proteins under acidic conditions and forms an intense pink proteineosin Y complex with an absorbance maximum of 535-545nm. The color produced from this reaction is stable and its absorbance increases proportionally over a wide range of protein concentrations (standard assay, 50-1000μg/ml of BSA; microassay, 1.25-50μg/ml of BSA). The eosin Y method can be performed at room temperature without further incubation after vortex mixing. The assay has advantages such as (i) simple procedures, (ii) stable dye-protein color with no precipitation of dye-protein complex for at least 72h, (iii) good reproducibility and (iv) rapid reaction. The characterization of the eosin Y method extends its best application to a routine protein determination in solution.

Fluorometric determination of gastric acidity with 2-fluorenecarboxaldehyde hydrazone

Fluorenecarboxaldehyde hydrazone (FCH) was synthesized as a new fluorescent reagent for the determination of acidity. FCH had no fluorescence in itself, however, it strongly fluoresced under acidic conditions at excitation maximum of 392 nm and emission maximum of 447 nm, respectively. It showed good correlation with pH in the range from pH 0.60 to pH 3.60 in strong acids. As an application, the acidity of gastric juice in rats and humans was determined. In comparison with pH-metry, the acidity measured by the developed method showed generally increased values of about 0.7–1.3 unit and 0.8–1.2 unit in rats and humans, respectively. However, these results had statistically close correlation with those of pH-metry and the correlation coefficients were 0.806 and 0.887 in rats and humans between two methods.

A Fast and Sensitive Coomassie Blue Staining for Proteins in Polyacryl-Amide Gels using Ion-Pairing Agent

Abstract Here we describe a fast and sensitive Coomassie blue staining method for detection of proteins in polyacrylamide gels using an ion-pairing agent, tetraoctylammonium bromide (TOA). To increase sensitivity, sodium dodecyl sulfate (SDS) in an electrophoresed gel was removed by washing with water for 5 min, and then the gel was stained in the mixed solution of 0.25% Coomassie brilliant blue R-250 (CB), and 1% TOA (mixed up at a 10 : 0.6 volume ratio). TOA inhibits the binding of CB to gel matrix by forming an ion-pair complex, thus not only increases the staining of CB but also helps make background destaining easy. This staining method reduces the staining and destaining times to 50 min and increases the detection limit up to 25 ng of bovine serum albumin (BSA).

Improved phosphotyrosine analysis by TLC and HPLC

We describe here the conditions of thin layer chromatography (TLC) and high pressure liquid chromatography (HPLC) to improve the analytical method of phosphotyrosine (P-Tyr) in biological sample. TLC was performed on silica plate with the mixture of propanol and water (2.1∶1 v/v) as a mobile phase and R1 values were 0.42, 0.39 and 0.33 for phosphotyrosine, phosphothreonine and phosphoserine, respectively. HPLC was performed on NH2 column with a mobile phase of potassium biphosphate solution by UV detection at 192 nm. The optimum condition of HPLC was obtained at 0.01 M, pH 4.5 with a clear separation within 12 min. These procedures have been applied to the analysis of phosphotyrosine obtained from tyrosine-phosphorylated enolase. Both TLC and HPLC methods were suitable to analyze tyrosine-phosphorylated protein without being affected by contaminants from hydrolysates.

Determination of total glycyrrhetic acid in Glycyrrhizae Radix by second derivative UV spectrometry

Second derivative (D2) spectrometry using ion-pair extraction technique was developed for the determination of total glycyrrhetic acid (GA) in Glycyrrhizae Radix. Glycyrrhizin (G) obtained from Glycyrrhizae Radix was hydrolyzed into GA in 2 N-HCl and methanol (1∶1) and extracted from aqueous phase in the form of an ion-pair complex with tetrapentylammonium bromide (TPA) as a counter ion. Maximum D2 amplitude (Z value) was obrained when 1000-fold or greater molar ratio of TPA was used at pH 11. Reaction time, temerature and ionic strength did not affect ion-pair formation. Dichloromethane was an effective extraction solvent of the ion-pair complex. The linearity of standard curve of ion-pair GA was obtained in the range of 4–120 μg/ml as GA. Assayed contents of GA in dry powder by D2 UV spectrometry and HPLC method were 5.31±0.04% and 5.20±0.008%, respectively.