Hello,
I am trying to optimize my primary antibody dilutions for semi-quantitative western blot.To perform that, I decided to try increasing my dilutions gradually, but my question is : where do I stop ?
I am worried that at some point, I will litteraly not have enough antibody for all the proteins to be recognized, thus biasing my interpretation.
You can find attached an example of WB I did.
Technically speaking, I put 6ug of proteins per well. I'm using nitrocellulose membrane, blocked with Casein blocking buffer 1X from Sigma. The primary antibody was incubated for 1 hour and is the mouse@pan-alpha-tubulin clone DM1A (from Sigma, T9026) at various dilutions (see attached) diluted in TBS-Tween (0.1%) + BSA (0.1%). Secondary antibody was incubated for 1 hour and is an HRP-coupled goat anti-mouse antibody diluted at 1/20000 in TBS-Tween (0.1%) + BSA (0.1%). Revelation of antibody labeling was made using ECL Western Blotting detection Reagent from Biorad (3 minutes incubation). Readout was performed using a chemiluminescent ChemiDoc Imaging system.
Thank you very much for your help,
Best,
Dear colleague:
In order to set your antibody you must put increasign quantities of your protein sample in the same membrane (i.e. 5, 10, 20, 40 ug) and then perform the western. Considering what we can see in your picture, your antibody is supergood, so I recomend you to use it at 1/50000 dilution. In the final picture you must see an increase in the signal in a 2x factor (because you are dupicating the amount of protein). You must do the same for your loading control. Next, you must plot the signal (Y axis) v/s amount of protein (X axis) for each protein and compare the slope of the linear regression. The slope must be the closest to 1. If you want to normalize using that loading control, both slopes must be very similar.
Wish you luck!!!!!!!
If the goal is to quantify the amount of tubulin in the sample, you should find out what combination of amounts of protein sample and antibody dilution will give the largest signal that does not saturate the detection capability of the device or film used for the quantitation. In other words, you should establish the upper end of the dynamic range. There will not be a single answer, so you just have to choose one pair. Having done that, you will have to vary the protein sample amount at a fixed antibody amount to establish the lower end of the dynamic range. This series of protein concentrations will constitute your standard curve. CCD cameras have a larger dynamic range and are more quantitative than film, so use the CCD camera if you can.
Blot looks really clean, you may choose 1:15000 and vary secondary.
Coming to your question : You will get very thin line at 1:60,000 that looks like maximum and it seems that your signal will disappear at 1:100,000
Thank you very much for your answers, all of you !
As you advised me to do, I'll stick with one or two dilutions and proceed with trying various protein quantities !
Thanks again, I really appreciate your help :)
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