On the other hand, low SDS concentration may not denature proteases and may not provide sufficient negative charge to proteins. These drawbacks result in fainter and less clear bands in Western blot experiments, due to protein degradation and less uniform migration velocity. Therefore, in follow-up experiments we attempted to stabilize native dimers using crosslinking agents to detect them in standard SDS-PAGE. The amino group-specific bifunctional crosslinker disuccinimidyl suberate was earlier used by two groups to improve the detection of Hv1 dimers in Hv1 transfected cell lines. Treating granulocytes with DSS before cell lysis, efficiently increased the detection of the dimeric Hv1 form in standard reducing PAGE. Additionally, the presence of Hv1 homodimers in all major types of human leukocytes and in differentiated PLB-985 granulocytes could be confirmed using DSS. Because monomeric and dimeric Hv1 forms support kinetically different voltage-gated proton currents, it was suggested that the transition between the two forms could lead to some of the proton current phenotype changes observed in granulocytes during phox activation.. Importantly, however, pretreating cells with a supramaximal phox activating dose of PMA before DSS addition did not alter its crosslinking potential in granulocytes or in differentiated PLB-985 cells. Although Hv1 dimers do not seem to decompose upon cell activation, disulfide formation has been reported to participate in redox regulation of different ion channels. We used Nethylmaleimide and N,N9- dimaleimide to explore the possible influence of redox changes during the PMA-induced oxidative burst on disulfide formation. Both NEM and PDM react covalently with reduced cysteine residues, but while PDM is a homobifunctional crosslinker and mimics disulfide formation, NEM prevents it. To get a snapshot on the redox state of the cysteines possibly involved in Hv1 dimerization, the thiol reactive compounds were added after the cells were placed on ice. PDM could drive dimer formation near to completion during the 40 min incubation. On the other hand, NEM almost completely prevented the detection of the dimer independently of PMA preactivation. These results indicate that although Atropine sulfate certain cysteines in the apposed Hv1 monomers become vicinal, they are mainly in reduced state. Massive oxidation of the apposed cysteines is unlikely even in PMA-activated cells, as the action of PDM and NEM on dimer formation was hardly affected by PMA treatment. Importantly, PMA treatment did not alter the amount of Hv1 protein in granulocytes. The detected Hv1 density in WBs from PMA-treated samples was 96610% of that from resting cells. It is theoretically possible that not monomer and dimer, but a higher-order multimer is the dominant native confirmation of Hv1 in granulocytes. We reasoned that we should be able to detect such multimers as clear and dominant band if we apply more intense crosslinking or if we solubilize cells with nonionic detergent after DSS crosslinking. In all cases, however, massive labeling was detected only around 70 kDa and in the stacking region, even if monomers became completely undetectable. Labeling in the stacking region may result form non-specific interactions and/or network like crosslinking of macromolecular complexes. Therefore, if higher-order multimers are present in granulocytes their occurrence is much less likely than that of dimers or interactions with other proteins. Among granulocytes PLB-985 cells displayed the most pronounced granular pattern, but in none of the tested leukocyte types localized Hv1 dominantly to the cell surface. Weak or no signal was present over the nucleus in mature cells. To verify the results obtained with confocal microscopy, WBs were performed on subcellular fractions of resting neutrophils. Confirming the results from confocal slices, significant Hv1 signal could be detected in fractions of both granule and plasma membrane Mechlorethamine hydrochloride origin.