Marc Moeremans

Sensitive colloidal metal (gold or silver) staining of protein blots on nitrocellulose membranes

A sensitive staining method for protein blots on nitrocellulose membranes is described and compared with commonly used dye staining methods. It uses colloidal metal sols (gold or silver) stabilized with Tween 20 and adjusted to pH 3. It is based on the selective high-affinity binding of colloidal metal particles to the proteins and produces a red-purplish color (gold) or dark grey (silver). The sensitivity of this new staining method is in the same range as silver staining of polyacrylamide gels and matches the sensitivity of overlay assays. It will therefore be a useful tool for correlating the position of bands or spots of proteins detected with overlay assays with the complete electropherogram in a duplicate protein blot.

Virological and immunological analysis of a triple combination pilot study with loviride, lamivudine and zidovudine in HIV-1-infected patients

Objective:To compare two antiretroviral regimens, loviride plus lamivudine (3TC) plus zidovudine (ZDV) (triple combination) and loviride plus ZDV (double combination) in terms of pharmacokinetic interactions, tolerability, safety, and immunological and virological efficacy. Study design:An open, case-controlled, pharmacokinetic and 24-week continuous treatment pilot study. Patients:Twenty p24 antigen-positive patients, 10 per treatment group, were matched according to p24 antigenaemia less or more than l00 pg, CD4 count less or more than 150×106/l, and gender. Eight out of 10 cases and seven out of 10 controls had received previous antiretroviral therapy. Results:No clinically relevant pharmacokinetic interactions were observed. Both treatment combinations were well tolerated. Median absolute and percentage CD4 count increases above baseline were more pronounced in the triple combination arm than in the double combination arm. Median p24-antigen and plasma viraemia level decreases below baseline were more pronounced in the triple combination arm. The M184I/V mutation was detected in all plasma samples of triple combination patients examined at week 12. Mutations conferring resistance to loviride and ZDV were found in a significant subset of patients in both treatment arms. Conclusions:Both combination regimens have an excellent safety/tolerability profile, but a higher level of in vivo efficacy is achieved by the triple combination, despite genotypic changes conferring resistance to one or all of these agents. The conclusions drawn are limited by small population size and the heterogenous pretreatment history. However, they support the validity of and strongly encourage a rationally designed multidrug combination approach to HIV therapy.

FerriDye: Colloidal iron binding followed by Perls' reaction for the staining of proteins transferred from sodium dodecyl sulfate gels to nitrocellulose and positively charged nylon membranes

A staining method for proteins on (positively charged) nylon and nitrocellulose membranes is described. The two-step method uses cationic cacodylate iron colloid which is substituted with Tween 20 at an OD460 nm = 0.5, followed by Perls reaction with acid potassium ferrocyanide. It stains transferred proteins deep blue with low background. The sensitivity is intermediate between that of conventional stains and AuroDye, the colloidal gold stain. This is the first sensitive staining method for proteins transferred on (positively charged) nylon membranes. These membranes have documented advantages in immunoblotting. It will therefore be a useful tool for correlating the position of bands or spots of proteins detected with overlay assays with the complete electropherogram in a duplicate protein blot.

Sequential immunostaining (gold/silver) and complete protein staining (AuroDye) on Western blots

A double staining method is described which combines immunodetection with sensitive staining of the complete electropherogram on the same membrane. The method is based on the use of Tween 20 as blocking agent, and uses immunogold/silver staining of specific antigens and gold staining of the overall protein pattern with AuroDye. This double staining makes possible the exact location of an immunodetected band within a complex protein pattern.

Colloidal Gold for the Detection of Proteins on Blots and Immunoblots

Immunoblotting techniques, in which electrophoretically separated proteins are transferred to the surface of nitrocellulose or nylon membranes (Western blots), are becoming increasingly popular in the analysis of protein systems. Transfer techniques in protein blotting have been covered in this series ( Chapter 20 in vol. 1, and Chapters 28 and 29 in this volume). In this chapter we will describe the use of the colloidal gold marking system for the detection of proteins on blots.

Dynamics Of Cell Components Investigated With Nanovid Microscopy

A new technique for the visualization of small molecular components in living cells is presented. It is based on the exploitation, by means of video techniques of the slight diffraction power of small metal particles (20-40 nm) in the light microscope. We will show that colloidal gold probes, coupled to selective antibodies or charge modulating molecules allow to follow and quantify dynamic processes in endocytosis, Saltatory Motion and membrane mobility.