What kind of antibodies to use? Conjugated vs. unconjugated? - (Sep/29/2006 )
Sorry if this seems to be a simple question but how do we know what kind of antibodies to use? I mean what is the difference between conjugated and unconjugated and how do we know which one to use? So, if we have an unconjugated primary antibody do we need to use an unconjugated secondary? Also, as far as dilutions go for the antibody, the information sheet says 1:500 for ascites, 1:1000 for purified Ig, and 1:500-1:1000 for polysera. My antibody is monoclonal unconjugated so which one do I follow? What does ascites mean? Does polysera mean polyclonal? There's so much I don't understand.
conjugated means labeld with f.i. fluorescent dye, ferromagnetic beads, agarose bead and so on, depends on your method; for Western blot most take unconjugated primary Ab, secondary labeled with AP or HRP; in your case use dilution of purified IgG; ascites IgG should be especially for ethical reasons not used; sera dilutions are for unpurified polyclonals that means different plasma cells produce antibodies against the same antigene but against different epitopes; monoclonals atre therefore more epitope-specific than polyclonals means practically, monoclonals give often (but not always) a better signal/noise ratio than polyclonals
If you have a monoclonal antibody you will want to get a secondary that is anti mouse IgG (H+L) conjugated to HRP enzyme or Alkaline Phosphatase, but most people use HRP for western. I dilute all my primary antibodies in 2% BSA/TBST, but everyone uses a different variation I think. I found that usually works well, and if there is too much background I up the BSA to 5%. I use HRP secondary from Jackson ImmunoResearch, and I like it a lot at 1/20,000 dilution = 40ng/ml.
Thanks very much for explaining to me! I did use HRP anti-mouse as my secondary antibody, it's just that my western is not working and I am trying to troubleshoot what could be the possible reasons. I guess it is not my choice of antibodies, although I did leave the secondary antibody out at room temperature for about an hour. =T
That won't matter. What concentration of primary antibody did you use? And what kind and amount of samples did you run on the gel?
I ran 9 samples of protein lysates extracted from ovarian tumors. There were concentrations on the tubes but I was told they were inaccurate so I just estimated how much to load based on a stained sds gel of the proteins. I loaded anywhere from 10ul to 30ul of protein on the lanes. I used a 1:500 dilution of my primary and 1:8000 dilution of my secondary since I was told it was a very good secondary antibody. I had done the transfer from gel to membrane overnight at 50V and had gotten an incomplete transfer but I at least saw the ladder dye (pre-stained ladder) had transferred over to the blot a bit. This time I re-did the whole thing transferring at 100V at room temperature for an hour and I saw absolutely nothing on my blot, not even the ladder. I also made my transfer buffer without SDS this time. My protein is 18kd so it shouldn't take that long to transfer. I have no idea what is wrong and I've been in lab till 9pm the whole week! what is going on???
You should try lower voltages, especially since your protein is so small. I do mine at 10V in a semi-dry unit for an hour. Did you see dye on the filter paper underneath the membrane? Was the protein still in the gel when you tried 100V for 1 hr? Did you get a lot of background or just no bands? Do you have methanol in your transfer buffer?
It's almost 10 and I'm still in lab and this time I stained my membrane with ponceau and I saw the my protein has transferred over! However, I also stained the gel and see some protein left over as well. And yes, the ladder dye did go through the membrane, so does that mean my protein passed over my membrane? but there is still some left in the gel so doesn't that mean it was an incomplete transfer again? Anyhow at least I have something I'll try to probe it and hopefully get something. One more thing though, is ecl solution still good after left out in room temperature for one day? thank you so much WAstate for guiding me through this!
No problem! I'm not an expert by any means, but I have been doing a LOT of westerns in the last 6 months and have made and learned from my mistakes;) I'm sure the ECL is still good. They tell you to bring it to RT anyhow before you use it, so one day is not going to kill it. I have seen people on the forum say you can add peroxide to the buffer if you need more signal, 1ul/5ml. I haven't needed to do it yet though.
Your transfers, as I understand, will never be truly complete because you're dealing with such a range of sizes. If you know you are looking for a smaller sized protein, then I would keep the transfers shorter than if you know you are looking for a larger protein. If your ladder went through the membrane, then I would say that your protein definitely did too, but if you see some on the membrane at the right size then it's possible that you will see results with your antibody, but you may want to increase the concentration of your primary just in case the antigen is in low supply. If you don't get results, then the 18kD protein probably went through the membrane and you should just work on your transfer time and voltage. Are you using a wet transfer unit or a semi-dry?
I hope you're not in the lab so late again!
Yes I was in lab until 12 midnight yesterday and still in lab now. Honestly, I'm not even sure my protein was on the membrane to begin with because I couldn't make out my ladder that well, I only saw distinct protein bands that were larger in size on top of the gel and moved on hoping I had something there. I tried developing the film and saw nothing at all. Could it be I'm not developing the film long enough? because I don't even see the outline of the membrane itself, even if there was no protein shouldn't I still see the outline of the membrane blot paper on the film? And can we re-use the same film over after we develop it if we haven't exposed it to light? the strange thing is I stained my gel and saw that the protein of my size was still left on the gel itself eventhough the ladder had passed through the membrane. So does that mean my protein is not transferring over or is it slipping through? I can't even pinpoint where the problem is!