The following protocols have been tested with this antibody:
Profilin antibody is provided as an affinity purified rabbit polyclonal antibody. The antibody has been raised against purified human profilin and it is predicted to have broad species cross reactivity. The positive control protein consists of an extract of bovine thymus that identifies a characteristic profilin band at 14 kDa. The control protein (bovine thymus extract, Cat. # EXT03) is supplied as a lyophilized powder; this should be reconstituted in 500 μl of cold Milli-Q water to 2 mg/ml. After addition of water the protein should be left at room temperature for 5 minutes with occasional pipetting up and down to make sure that the protein has completely dissolved into solution, it can then be stored at -20°C. We recommend running 10 μg of positive control protein in Western blots.
The profilin antibody (50 μg) is supplied as a lyophilized powder. It is recommended to resuspend the powder in 200 μl of 30% glycerol/water. This gives a concentration of 250 μg/ml in PBS plus 30% glycerol with 0.01% chlorhexidine for preservative.
Figure 1: Western blot probed with profilin antibody. Lane 1; 100 ng profilin protein, Lane 2; 10 μg thymus extract protein, Lane 3; 10 μg of platelet extract protein. Primary antibody used at 1:1000 dilution, secondary antibody used at 1/50 000 dilution anti-rabbit HRP, Supersignal West Dura™ (Pierce Co.) detection method with film exposure for 30 seconds.
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|Hernández-Deviez, Delia J. et al.||ARNO and ARF6 regulate axonal elongation and branching through downstream activation of phosphatidylinositol 4-phosphate 5-kinase alpha||Molecular biology of the cell||2004||ISSN 1059--1524|
|Innocenti, Metello et al.||Abi1 is essential for the formation and activation of a WAVE2 signalling complex||Nature cell biology||2004||ISSN 1465--7392|
|Tang, Dale D. et al.||Role of Crk-Associated Substrate in the Regulation of Vascular Smooth Muscle Contraction||Hypertension||2003||ISSN 1524-4563|
|Tang, Dale D. et al.||Downregulation of profilin with antisense oligodeoxynucleotides inhibits force development during stimulation of smooth muscle||American journal of physiology. Heart and circulatory physiology||2003||ISSN 0363--6135|
Question 1: What is the specificity of the profilin antibody?
Answer 1: The profilin antibody (Cat. # APUF01) will specifically react with profilin of mouse, rat, and human origin, there are no other bands on the western blots.
Question 2: What epitopes on profilin does this antibody recognize?
Answer 2: The profilin antibody (Cat. # APUF01) is a polyclonal antibody and so recognizes multiple epitopes on human profilin as it was raised against the entire purified human profilin protein.
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