Showing papers on "Electroblotting published in 2008"

Labelling of proteins with 2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid and lanthanides and detection by ICP-MS

Abstract: The labelling of two different proteins (bovine serum albumin, hen egg white lysozyme) with the commercially available chelating compound 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (p-SCN-Bn-DOTA) has been investigated. The assay described here has been optimised for the application to detect proteins labelled by stable isotopes of Eu, Tb and Ho and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Detection has been performed by laser ablation ICP-MS after electroblotting of the target proteins onto NC membranes. A range of total protein amounts from 0.015 pmol (BSA) to 105 pmol (lysozyme) has been covered. A calibration was performed for BSA in the range from 0.015 to 15 pmol and a limit of detection below 15 fmol can be estimated. For lysozyme integrated sensitivities of more than 107 cps pmol−1 of protein have been realized. The conditions, once optimised for labelling with Eu, have been applied for other lanthanides (Tb, Ho), too. ESI-MS of the intact and the tryptic digested lysozyme has been used as to provide a better understanding of the reaction chemistry and efficiency. The procedure described here looks promising to develop multielement labelling strategies (multiplexing) for ICP-MS applications in quantitative proteomics.

Protein Blotting: Immunoblotting

Abstract: Immunoblotting (also referred to as Western blotting) uses antibodies to probe for a specific protein in a sample bound to a membrane. Typically, a protein sample is first size separated via electrophoresis (e.g., SDS PAGE). However, antibodies used for specific protein detection are restricted by the polyacrylamide gel and, to make the separated proteins accessible, the proteins need to be moved out of the gel and bound to a rectangular sheet of PVDF or nitrocellulose membrane. In this second step, the membrane, cut to the same dimensions of the SDS gel (e.g., 10 x 10 cm), is then laid on the gel surface. The gel and membrane sandwich is then positioned in specialized blotting equipment that electrophoretically transfers the negatively charged proteins from the gel onto the membrane. The nitrocellulose or PVDF membrane binds the proteins as they move out of the gel, producing an exact replica, on the membrane surface, of the original protein gel separation. The proteins bind with high capacity and, in contrast to the polyacrylamide gel, are freely accessible to antibody reagents. The membrane is then blocked to prevent any nonspecific protein binding and visualized by specific antibodies to detect the presence or absence of a particular protein. For routine quantitation of a protein, the SDS PAGE separation is not always needed, and whole cell lysates or other complex mixtures are bound directly to the membrane for analysis using slot or dot blotting. Applications of immunoblotting are many, and include antibody characterization, diagnostics, gene expression and post translational modification analysis. Keywords: Western blotting; protein blotting; slot blot; dot blot; peroxidase; alkaline phosphatase; chromogenic; chemiluminescence; nitrocellulose; PVDF; TMB; DAB

Detection of proteins on blot transfer membranes.

Abstract: Staining of blot transfer membranes permits visualization of proteins and allows the extent of transfer to be monitored. In the protocols described in this unit, proteins are stained after electroblotting from one-dimensional or two-dimensional polyacrylamide gels to blot membranes such as polyvinylidene difluoride (PVDF), nitrocellulose, or nylon membranes. Protocols are provided for the use of six general protein stains: Amido black, Coomassie blue, Ponceau S, colloidal gold, colloidal silver, and India ink. In addition, the fluorescent stains fluorescamine and IAEDANS, which covalently react with bound proteins, are described. Approximate detection limits for each nonfluorescent stain are indicated along with membrane compatibilities. Curr. Protoc. Immunol. 83:8.10B.1-8.10B.6. © 2008 by John Wiley & Sons, Inc. Keywords: nylon; nitrocellulose; PVDF; protein/blotting; stain; total protein; fluorescence

Polymeric sorbent sheets as ion reservoirs for electroblotting

Abstract: Electroblotting for the transfer of electrophoretically separated species from a gel to a transfer membrane is performed in a semi-dry format with sheets of absorbent polyester, polyester/cellulose blend, or polyurethane-coated cellulose wetted with buffer solution in place of the traditional buffer-wetted filter paper. The result is effective electroblotting at a lower electric current level than that obtained with filter paper and thereby less resistance heating of the gel, the transfer membrane, and the species being transferred.

Array-based biomolecule analysis method

Abstract: An array (100) of macromolecules (the primary array), typically proteins is generated by 2D electrophoresis, for example, and subsequently transferred to a support membrane (102) by electroblotting or the like. An image of the primary array is captured (202) and the coordinates of the various macromolecular spots in the primary array are determined (402). The next step of the process is to print a secondary (or micro) array of one or more reagents or chemicals onto one or more spots/coordinates of the primary array with a pico-litre (pl) dispenser (702). If the macromolecules are proteins, the reagents may be enzymes such as Trypsin or GluC. Use of two different enzymes deposited onto different coordinates on the same spot will cleave the protein at different amino acid sites and, when the spot is analysed in a MALDI-TOP mass spectrometer, will provide increased coverage or matching of peptides in the protein.

Sequencing of Amino Acids at N-terminus of Recombinant Antibody

Abstract: Objective To develop a method for sequencing of amino acids at N-terminus of recombinant antibody with pyrog- lutamate blockage. Methods The pyroglutamate at N-terminus of recombinant human anti-rabies virus McAb was removed at high temperature by using thermal-stable Pfu pyroglutamate aminopeptidase, based on which the McAb was identified by reduced SDS- PAGE and electroblotting and analyzed for sequence at N-terminus. The result was compared with that of McAb deblocked on PVDF membrane after electroblotting. Results The pyroglutamate at N-terminus of recombinant human anti-rabies virus McAb was re- moved effectively by the developed method, and the N-terminal amino acids of McAb after deblocking was sequenced successfully. However, the pyroglutamate at N-terminus of McAb deblocked on PVDF membrane after electroblotting could not be removed effec- tively, and the N-terminal amino acids could not be sequenced. Conclusion The developed method was suitable for the N-terminal amino acid sequencing of McAb with pyroglutamate blockage.

Automatic transfer of polyacrylamide gels to protein chip plates for protein electroblotting

Abstract: Protein chips are useful tools for profiling proteins and analyzing protein-protein interactions and post-translational modifications. In previous work, we developed a diamond-like carbon-coated stainless steel plate (DLC plate) as a novel protein chip plate. Gel-resolved proteins can be covalently immobilized on the surface of the DLC plate by electroblotting to produce a high-density protein chip. The proteins can then be identified by matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF MS) using the plates. The interactions of the immobilized proteins with other proteins, and their post-translational modifications, can also be analyzed. However, to improve the efficiency and reproducibility of analyses using DLC plates, it is important to automate these analytical processes. Therefore, we developed a system for automatically transferring gels from the gel electrophoresis glass plates to the DLC plates for electroblotting. This is an essential first step toward complete automation of the production of high-density protein chips for immobilizing gel-resolved proteins.