Quantitative filtration-blotting of protein in the presence of sodium dodecyl sulfate and its use for protein assay

Nishibu, Takahiro; Hirayasu, Kazunari; Tanaka, Takumi; Takeda, Yasuhiro; Kobayashi, Yasukazu

doi:10.1016/s0003-2697(03)00255-0

Cited by 9 publications

(5 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The results of this analysis show that PI-PLC treatment does not reduce the actual amount of PrP C present in incubated samples ( Figure 1B), whereas the Western blot signal for PI-PLC treated samples of purified PrP C are reduced compared to untreated samples ( Figure 1C, lanes 1-4). Second, we treated samples containing recombinant hamster PrP expressed in E. coli (and therefore lacking a GPI anchor) (InPro Biotechnology, South San Francisco, CA) with PI-PLC, and observed no reduction in Western blot signal ( Figure 1C, lanes [5][6]. This result shows that enzymatic treatment does not reduce Western signal intensity if the substrate protein lacks a GPI-anchor.…”

mentioning

confidence: 91%

“…We next tested the possibility that PI-PLC treated proteins might either: (1) be retarded in migrating out of polyacrylamide gels during Western transfer, or (2) fail to denature completely during Western transfer, resulting in lack of epitope exposure. SDS-denatured samples containing purified PrP C molecules were directly applied onto PVDF membranes using a modification of a well-characterized slot blot method [5], and the membranes were probed with two different anti-PrP antibodies that recognize spatially distinct epitopes [6]. The results of this experiment show that PI-PLC treatment reduced the slot blot signals with both antibodies ( Figure 1D).…”

mentioning

confidence: 96%

See 1 more Smart Citation

Immunodetection of glycophosphatidylinositol-anchored proteins following treatment with phospholipase C

Nishina

Supattapone

2007

Analytical Biochemistry

View full text Add to dashboard Cite

Many important regulatory proteins are attached to the extracellular leaflet of the plasma membrane by a glycophosphatidylinositol (GPI) anchor [1]. One of the most common methods used to determine whether a protein contains a GPI anchor involves treating membranes with the enzyme phosphatidylinositol-specific phospholipase C (PI-PLC), followed by detection of the "released" candidate protein into the supernatant fraction by traditional Western blotting [2]. Here, we report that the amounts of two representative GPI-anchored proteins, Thy-1 and the cellular prion protein (PrP C) [3], detected by Western blotting decreased >95% following PI-PLC treatment. The diminished signals appear to be caused by failure of the PI-PLC treated proteins to adhere to PVDF or nitrocellulose membranes, and could lead to false negative results in PI-PLC release assays. Detection of PI-PLC treated PrP C molecules was successfully achieved by (1) slot blotting in the absence of SDS onto negatively charged Nylon membranes and (2) in-gel immunostaining.

show abstract

mentioning

confidence: 91%

mentioning

confidence: 96%

Immunodetection of glycophosphatidylinositol-anchored proteins following treatment with phospholipase C

Nishina

Supattapone

2007

Analytical Biochemistry

View full text Add to dashboard Cite

show abstract

“…Amounts of HSA protein of decreasing concentrations (500 ng to 3.9 ng) were loaded into the 12% SDS-polyacrylamide gel with the protein marker (LMW-SDS Marker Kit, GE) and separated under a constant voltage of 200 V for 45 min. The gel was then fixed and stained with dye for 30 min and analyzed with a Typhoon Trio Imager (GE) with excitation and emission at 457 nm and ∼620 nm, respectively …”

Section: Methodsmentioning

confidence: 99%

“…The gel was then fixed and stained with dye for 30 min and analyzed with a Typhoon Trio Imager (GE) with excitation and emission at 457 nm and ∼620 nm, respectively. 35 The Cellular Uptake of Europium Complexes. To measure the intracellular concentration of the complex, 1 Â 105 cells were plated in each well and incubated with the complex with different concentrations (0.01, 0.025, 0.05, 0.1, and 0.2 mM).…”

Section: Articlementioning

confidence: 99%

In Vitro Imaging and Human Serum Albumin Responsive Dimeric Lanthanide DO3A Complex

Fung

Yeung

et al. 2011

Inorg. Chem.

View full text Add to dashboard Cite

Two series of dimeric DO3A (1,4,7,10-tetraazacyclodecane-1,4,7-triacetate) lanthanide complexes (LnL(1)-LnL(2), Ln = Eu, Gd, and Tb) have been synthesized with two different bridged chromophores. The X-ray structures of dimeric LnL(1) (Ln = Gd and Tb) complexes show that each metal ion has nine coordination numbers with eight directly bound donor atoms of the ligand and one oxygen donor from the water molecule. Photophysical measurements indicate that the bridged antenna in LnL(2) gives a higher efficiency than that of LnL(1) and is responsive to the protein Human Serum Albumin (HSA), giving an f-f luminescence signal enhancement with a binding constant log K = 4.84. In vitro imaging of EuL(1) and EuL(2) in HeLa cells has been recorded, and EuL(2) has demonstrated a higher rate of cellular uptake and low cytotoxicity (IC(50) = 3 mM).

show abstract

“…A solid-phase protein assay provides a good alternative. − With the protein immobilized on a solid surface such as a nitrocellulose membrane or a PVDF membrane, interfering substances could be washed away. Thus, appropriate staining techniques are required to detect proteins in the nanogram to microgram range.…”

Section: Introductionmentioning

confidence: 99%

Microwave-Enhanced Ink Staining for Fast and Sensitive Protein Quantification in Proteomic Studies

Cheng

Liu

2006

J. Proteome Res.

View full text Add to dashboard Cite

A novel microwave-enhanced ink staining method was developed for rapid and sensitive estimation of protein content in sample buffers containing chaotropes, dyes, detergents, and reducing agents. Dye-based Blue-Black ink was used to quantitatively visualize proteins spotted on a nitrocellulose membrane. The total staining time was greatly reduced to 3 min by brief exposure to microwave radiation. The stained membrane was washed with distilled water, baked in a microwave oven for complete desiccation, transparentized with mineral oil, and documented by a desktop scanner or densitometer. Only 1 microL of protein sample (protein solubilized in SDS-PAGE sample buffer or IEF rehydration buffer) was used for protein spotting. The novel solid-phase protein assay gives a 500-fold dynamic range from 19.5 to 10000 ng/microL and can be scaled up for high-throughput protein quantification analysis. The fast, sensitive and low-cost microwave-enhanced ink staining procedure is ideal for protein quantification in proteomic analysis.

show abstract

Quantitative filtration-blotting of protein in the presence of sodium dodecyl sulfate and its use for protein assay

Cited by 9 publications

References 21 publications

Immunodetection of glycophosphatidylinositol-anchored proteins following treatment with phospholipase C

Immunodetection of glycophosphatidylinositol-anchored proteins following treatment with phospholipase C

In Vitro Imaging and Human Serum Albumin Responsive Dimeric Lanthanide DO3A Complex

Microwave-Enhanced Ink Staining for Fast and Sensitive Protein Quantification in Proteomic Studies

Contact Info

Product

Resources

About