Maowei Ni

An improved silver stain for the visualization of lipopolysaccharides on polyacrylamide gels.

A sensitive, brief, and user‐friendly silver stain to meet the needs in high‐efficiency detection of lipopolysaccharides (LPS) on polyacrylamide gels is described. In this study, the most commonly used formaldehyde‐based LPS silver stain, which is potentially hazardous to the operator, is replaced by ascorbic acid (Vc) in alkaline sodium thiosulfate solution. It takes only about 35 min to complete all the protocol, with a detection limit of 4 ng of total LPS. The results indicate that this user‐friendly method could be a good choice for LPS visualization on polyacrylamide gels.

A novel targeted proteomics method for identification and relative quantitation of difference in nitration degree of OGDH between healthy and diabetic mouse

For analysis of nitration modification of α oxoglutarate dehydrogenase (α‐OGDH) induced by diabetes, a targeted proteomics strategy was developed through the use of Skyline. All peptides containing Y and W of the target proteins were nitrated in silico and output to produce parallel reaction monitoring (PRM) or SRM method for nitration analysis. A nitrated casein mixture was used as standard protein to assess the feasibility of this method. The results demonstrated the availability of this strategy for nitration identification, and subsequently this method was used to analyze the nitration of α‐OGDH from myocardial tissue extracts of diabetic mouse. The PRM method was primarily generated by Skyline for identification of the actual nitrated peptides from all possible nitrated peptides of α‐OGDH due to the complexity of α‐OGDH. The PRM‐based data were analyzed by SEQUEST, and transitions of the identified nitrated peptides were used to develop an SRM method for relative quantitation of nitration degree. The nitration degree of α‐OGDH for diabetic mouse is higher than that for control mouse, indicating that α‐OGDH of the diabetic mouse suffered from more intense oxidative damage. We believe that this approach for obtaining information regarding nitration will facilitate the study of other PTMs in complex mixtures.

Improved staining of phosphoproteins with high sensitivity in polyacrylamide gels using Stains-All

An improved Stains‐All (ISA) staining method for phosphoproteins in SDS‐PAGE was described. Down to 0.5–1 ng phosphoproteins (α‐casein, β‐casein, or phosvitin) can be successfully selectively detected by ISA stain, which is approximately 120‐fold higher than that of original Stains‐All stain, but is similar to that of commonly used Pro‐Q Diamond stain. Furthermore, unlike the original Stains‐All protocol that was time consuming and light unstable, ISA stain could be completed within 60 min without resorting to protect the gels from light during the whole staining procedure. According to the results, it is concluded that ISA stain is a rapid, sensitive, specific, and economic staining method for a broad application to the research of phosphoproteins.

Fluorescent staining of protein in sodium dodecyl sulfate polyacrylamide gels by salicylaldehyde azine.

As a noncovalent fluorescence probe, in this study, salicylaldehyde azine (SA) was introduced as a sensitive fluorescence‐based dye for detecting proteins both in 1D and 2D polyacrylamide electrophoresis gels. Down to 0.2 ng of single protein band could be detected within 1 h, which is similar to that of glutaraldehyde‐silver stain, but approximately four times higher than that of SYPRO Ruby fluorescent stain. Furthermore, comparative analysis of the MS compatibility of SA stain with SYPRO Ruby stain indicated that SA stain is compatible with the downstream of protein identification by LC‐MS/MS. Additionally, the probable mechanism of the SA stain was investigated by molecular docking. The results demonstrated that the interaction between SA and protein was mainly contributed by hydrogen bonding and hydrophobic forces.

Fluorescent staining of protein in SDS polyacrylamide gels by salicylaldehyde azine

As a noncovalent fluorescence probe, in this study, salicylaldehyde azine (SA) was introduced as a sensitive fluorescence‐based dye for detecting proteins both in 1D and 2D polyacrylamide electrophoresis gels. Down to 0.2 ng of single protein band could be detected within 1 h, which is similar to that of glutaraldehyde‐silver stain, but approximately four times higher than that of SYPRO Ruby fluorescent stain. Furthermore, comparative analysis of the MS compatibility of SA stain with SYPRO Ruby stain indicated that SA stain is compatible with the downstream of protein identification by LC‐MS/MS. Additionally, the probable mechanism of the SA stain was investigated by molecular docking. The results demonstrated that the interaction between SA and protein was mainly contributed by hydrogen bonding and hydrophobic forces.

Improved conditions for periodate/Schiff's base‐based fluorescent staining of glycoproteins with dansylhydrazine in SDS‐PAGE

An improved periodate/Schiffs base based fluorescent stain with dansylhydrazine (DH) for glycoproteins in 1D and 2D SDS‐PAGE was described. Down to 4–8 ng of glycoproteins can be selectively detected within 2 h, which is approximately 16‐fold higher than that of original protocol, but similar to that of Pro‐Q Emerald 488 stain (Invitrogen, Carlsbad, USA). Furthermore, subsequent study of deglycosylation, glycoprotein affinity isolation, and LC‐MS/MS analysis were performed to confirm the specificity of the improved method. As a result, improved DH stain may provide a new choice for selective, economic, MS compatible, and convenient visualization of gel‐separated glycoproteins.

A method for sensitive staining of DNA in polyacrylamide gels using basic fuchsin

BACKGROUND PAGE is a widely used analytical method to resolve components of a DNA mixture based on their size. Various DNA visualization methods including fluorescence, visible dye and silver have been used for the detection of gel-separated DNA, with each having different advantages and disadvantages in terms of sensitivity, safety and simplicity. RESULTS A fast and sensitive visible dye-based staining method for DNA in polyacrylamide gels using basic fuchsin (BF) is described. As low as 10-20 pg of DNA can be visualized within 10 min; the sensitivity is fourfold more sensitive than that of SYBR® Gold stain, the most sensitive commercial fluorescent probe, but similar to silver staining kit from GE Healthcare. In addition, the mechanism studies suggest that the interaction of BF with DNA is mainly contributed by non-intercalative binding mode. CONCLUSION By comprehensive studies of this visible dye-based protocol, we concluded that BF stain is a fast and sensitive method currently available for detecting DNA in polyacrylamide gels.

A new fluorescent quenching method for the determination of phosphoproteins by using calconcarboxylic acid

A fluorescent quenching detection method for phosphoproteins in SDS‐PAGE by using calconcarboxylic acid (CCA) was described. In this method, the fluorescence intensity of CCA was greatly increased with the presence of Al3+ in the gel background, while in zones where phosphoproteins are located this intensity was absent because of fluorescence quenching phenomenon through the formation of CCA‐Al3+‐phosphoprotein appended complex. Approximately 4–8 ng of phosphoproteins can be selectively detected within 1 h (1D SDS‐PAGE), which is similar to that of the most commonly used Pro‐Q Diamond stain. The specificity of this novel technique for phosphoproteins was confirmed by dephosphorylation, Western blot, and LC‐MS/MS analysis, respectively. Furthermore, to better understand the newly developed method, the detection mechanism of CCA stain was explored by fluorescent spectrometry. According to the results, it is believed that CCA stain may provide a new choice for selective, economical, MS compatible, and convenient visualization of gel‐separated phosphoproteins.

Fluorescent staining of protein in SDS polyacrylamide gels by salicylaldehyde azine: Proteomics and 2DE

As a noncovalent fluorescence probe, in this study, salicylaldehyde azine (SA) was introduced as a sensitive fluorescence‐based dye for detecting proteins both in 1D and 2D polyacrylamide electrophoresis gels. Down to 0.2 ng of single protein band could be detected within 1 h, which is similar to that of glutaraldehyde‐silver stain, but approximately four times higher than that of SYPRO Ruby fluorescent stain. Furthermore, comparative analysis of the MS compatibility of SA stain with SYPRO Ruby stain indicated that SA stain is compatible with the downstream of protein identification by LC‐MS/MS. Additionally, the probable mechanism of the SA stain was investigated by molecular docking. The results demonstrated that the interaction between SA and protein was mainly contributed by hydrogen bonding and hydrophobic forces.