SFPQ, Abcam, ab50935

Used for Western Blot and immunofluorescence detection of SFPQ protein.

Experimental details:

Western blot (HeLa cells) :
-primary antibodies dilution: 1:2500-5000 of SFPQ antibody in 5%milk/PBS-T (incubation time: 1hr at RT or ON at 4 degrees). For ON incubation I have routinely used 1-5000 dilution.

-secondary antibodies dilution: 1:10.000 of goat anti-rabbit/HRP antibodies in 5%milk/PBS-T (DAKO #P0448) (incubation time: 1hr at RT),

-chemiluminescent substrate: SuperSignal West Femto (Thermo Scientific #34096)

NB! Protein was detected between 100~130kDa band of PageRuler Prestained Protein Ladder (Thermo Scientific #26616). Predicted weight 76 kDa. Manufacturer claims that they detect protein 77 kDa. However, someone has submitted a review to the Abcam page and he/she also detects protein with larger size (~118 kDa). Importantly, this band is much weaker in the SFPQ siRNA treated cells (see Abcam review page for the results).

Immunofluorescence (IF):

Hela cells fixed with 4%PFA/PBS, 15 min at RT. For permeabilization I have used 70% EtOH.

-SFPQ antibody dilution: 1:500-1000 in 1% BSA/PBS (incubation time: 1hr at RT). Signal predominately in the nucleus.