I am trying to perform western blots of alpha synuclein (14 kDa), using wet transference 1h 15min at 85 V, but I get this weird "patchy" pattern, the band isn't a whole defined band. What could be the problem?
Yes , the band indeed migrates around 18 kD, but my concern is the appearance of the band, because its difficult to quantify!
Thanks
That´s what I´m suspecting!
1) I use nitrocellulose membranes
2) 3.03 g Tris, 14.4 g Glycine, 20% Methanol and ddH2O up to 1 liter
3) Wet transfer (tank blotting) 1h 15min at 85 V
4) I incubate with the primary antibody overnight at 4 °C and then 1 hour at RT (in TBS 5% skim milk). The secondary antibody goes 1 h at RT in TBS.
Thank you I appreciate any advice you can give me!
Are you using 0.22 or 0.45 pore size nitrocelullose? 0.22 maybe more suitable for your protein of interest. 85V for 75 min is kind of high to me. I usually use 200-250 mA for 60 min which is about 40-45V. Use 15%, 4-15%, or 4-20% fel may also give you a better result.
Thank you Edgar Dawkins! I did blot the same membrane for GAPDH and it was fine! I should try changing the voltage for the transference... Thanks!
Ru-Jeng Teng thank you! My membranes are 0.22 ! The gel in the photograph is a 12%, I will try at a lower voltage!
I did not notice the gel attached. I never used florescent-conjugated 2nd. But now I see it. Actually the bands are not too bad as compare to most of my fellow students and the intensity should be quantifiable by imageJ. .
I agree with Ru-Jeng. The bands did not look very bad. I always use secondary antibodies conjugated to IR dye of to be detected at 680 or 800nm with an end point LiCor Odyssey IR scanner. After image acquisition the image can be processed further by altering intensity, brightness/contrast etc. globally using the specific acceptable image acquisition and processing soft wares.
This way the band intensity will be enhanced and changing the image to grey scale (BW) will help you finding the bands in a better way to be analyzed using image J or other image analysis soft wares.
Firstly, the bands are not too bad at all. I have seen worse published in Nature! :-)
The two most common reasons for similar non uniform bands I have come across are:
1) tiny bubbles in the transfer solution due to excessive mixing. A quick de-gasing in a vacuum flask can eliminate those.
2) Incomplete lysis of sample, e.g. high content of genomic DNA. Easiest solution, assuming that you have enough volume, is using a 1ml syringe and pipetting up and down 3 times through a fine needle. Works wonders on resuspended bacteria, for example!
Good luck!
I had this same problem, using the same buffer composition, with the same nitrocellulose, and the same detection method. In contrast to what others think, properly quantifying them were a challenge.
What worked for me, as others have suggested, decrease the transfer voltage. Best of luck.
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