ACT | Actin (recombinant monoclonal, clone 14H4G8)
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Store lyophilized/reconstituted at -20°C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles. Please, remember to spin tubes briefly prior to opening them to avoid any losses that might occur from lyophilized material adhering to the cap or sides of the tubes.
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No confirmed exceptions from predicted reactivity are currently known
10 µg/well of total protein extracted freshly from Arabidopsis thaliana leaf tissue. All lanes shown are from different Arabidopsis thaliana leaf samples extracted simultaneously. Fresh leaf tissue was ground up directly in 1x Bolt LDS loading buffer (Thermo) and 200mM DTT and denatured at 80°C for 10 min. Samples were separated on 12% SDS-PAGE gel and transferred to nitrocellulose by wet transfer for 1hr at 100V. Blot was blocked with 5 % milk in TBST for 1h/RT with agitation. Blot was incubated with Agrisera Mouse anti-actin monoclonal (AS21 4615) at a dilution of 1:1000, in 5% milk with agitation at 1h RT. The blot was rinsed 3 times for 5 minutes in TBS-T with agitation. Then the membrane was incubated with secondary anti-mouse HRP (Agrisera AS09 627) at 1:10 000 dilution in milk for 1hr at room temp. The blots were washed as above and reaction was visualized using ECL reagent and following manufacture's recommendations. The actin band was visualized after 15 seconds of film exposure.
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