esiRNA Knockdown Efficiency Tested by Western Blotting

A valuable measure of the knock-down potency of any RNAi experiment is the reduction in protein level. Displayed below are the knock-down rates of  selected esiRNAs using quantitative western blot analysis (Odyssey, Li-COR). The time point for maximum knock-down rate for each protein can vary significantly, as it is depending on factors such as protein stability, turn-over rate or cell proliferation rates. The knock-down validation on protein level was performed at 72 hours post transfection of esiRNA in HeLa cells. To be able to assess knock-down rates, the expression levels of the protein of interest was compared to HeLa cells simultaneously transfected with Renilla Luciferase (negative control). Specific antibodies for the protein of interest were used for the quantitative western blot analysis.

Quantitative Western Blotting

Western blot analyses 72 hours after transfection of HeLa cells with the indicated esiRNAs. Lane 1 contains, a negative control, HeLa cells transfected with Renilla Luciferase esiRNA. Lane 2 includes HeLa cells transfected with the indicated esiRNA. Lysates were separated on SDS-PAGE and probed with the indicated primary antibody. For lanes 1 and 2, the individual esiRNA targets are shown in green while the loading controls are in red. The signal intensities were quantified using an Odyssey infrared imaging system (LI-COR) and normalized to the loading control signal (GAPDH or tubulin).

	AKT1	OCT4	BCL2
esiRNA	EHU083501	EMU081351	EHU135281
Antibody	P2482-.2ML	P0082-200UL	B3170-.2ML

	CAT	HSP90	PLK1
esiRNA	EHU048671	EHU114671	EHU051011
Antibody	C0979-.2ML	H1775-.2ML	P5998-200UL

	CAV1	CDK4	ABL1
esiRNA	EHU031251	EHU070511	EHU141291
Antibody	SAB4200216	C8218-.5ML	A5844-.2ML

	FAS	VCL	CAPN1
esiRNA	EHU060231	EHU088101	EHU032581
Antibody	F4424-.1ML	SAB4200080-200UL	C267-100UL

	KRAS	PTEN
esiRNA	EHU114431	EHU106441
Antibody	R3400-.1MG	P3487

The reduction in target expression was measured between the negative control RLUC and the sample treated with esiRNA as indicated.

Materials

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