Aphidicolin Large-Scale Synchronization of Rapidly Dividing Cell Monolayers and the Analysis of Total Histone and Histone Variant Biosynthesis during the S and G2 Phases of the HEp-2 Cell Cycle
✍ Scribed by Thomae G. Sourlingas; Kalliope E. Sekeri-Pataryas
- Publisher
- Elsevier Science
- Year
- 1996
- Tongue
- English
- Weight
- 77 KB
- Volume
- 234
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
show detectable cross-reactions for LPO till lane 15 silver-stained gels do not take up the silver stain and thus remain clear. This permits use of detection meth-(0.93 mg LPO). Two cross-reacting proteins at the Ç80-kDa region were detected in SMG extract (lane 9), pos-ods which finally involve enzyme-based color reactions.
We have also successfully attempted poststain blotting sibly representing LPO-like proteins (5). Immediate blotting (Fig. 2F) of a duplicate gel of lanes 9-16 (trans-of bovine catalase (Sigma) and hamster lacrimal 20-kDa protein (4) probed with their respective antisera. fer time 1 h 30 min; autorad exposure time 5 h) showed similar sensitivity of detection for LPO and LPO-like Cross-reactions for catalase (M r 60 kDa) were clearly detected even below 1 mg (data not shown). The gener-SMG proteins as seen in poststain blotting.
alization of the potential usefulness of this technique to the majority of Coomassie-stained proteins needs DISCUSSION further investigation. In the present study, poststain blot allowed long storage of gels in destaining solution, Our results show that poststain blotting, using 3fold longer transfer time and 3.5-fold longer autorad photography, and evaluation of the quality of electrophoretic separation before a decision was taken to exposure than immediate blotting, gives presentable results comparable with immediate blotting. However, transfer a gel. For samples available in small quantities, protein profile evaluation (by Coomassie or silver using a poststain gel of submandibular extract (dilutions as in Fig. 2A) and employing similar transfer and stain) after a single electrophoretic run could be followed by poststain blotting for immunodetection. The autorad exposure times as for immediate blotting gave unacceptable results for the poststain blot where auto-most useful application could be in Western blots of separations which are difficult to exactly reproduce, rad signals for the 24-kDa protein were discernible only at the highest concentration (data not shown). The 45-e.g., two-dimensional gels. In such cases, exact superimposition of the autoradiogram on the photograph or min and 90-min transfer times employed here for immediate blotting of 20.5-30 kDa SMG proteins and nitrocellulose replica of the stained gel profile would permit exact localization of cross-reacting proteins. LPO (80 kDa), respectively, are widely used transfer times for such M r proteins. Longer transfer time causes a flowthrough of transferred proteins through nitrocel-REFERENCES lulose. Although longer transfer times were employed 1. Gershoni, J. M., and Palade, G. E. (1983) Anal. Biochem. 131, in poststain blotting, we could detect no autorad signals 1-15. in subsequent nitrocellulose papers kept behind the 2. Laemmli, U. K. (1970) Nature 227, 680-685. original (not shown). The poststain transferred pro-