What kind of antibodies to use? Conjugated vs. unconjugated? - (Sep/29/2006 )
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!
What I'm going to guess is that you're not transferring any of the proteins completely, and the ones that do transfer go through the membrane. I don't think you can reuse your film after it has been through the developer. You don't always see the outline of the membrane on film. I'd say that if you have to expose the film for over 30 minutes to one hour, there is not enough signal to detect in the first place. For 18kD I would use 0.2uM PVDF. Make sure you have 20% MeOH in your transfer buffer, and that you hydrate the membrane with 100% MeOH before transferring. PVDF will capture more protein than nitrocellulose. Are you using a wet or semi-dry transfer system? If you only saw larger distinct protein bands, then your protein definitely went through the membrane.
I'm using a wet transfer system, and we don't have PVDF unfortunately. I'd like to think that my proteins went through the membrane since I only see the larger protein bands on the nitrocellulose paper but I stained my gel and most of the protein is still remaining on my gel so it makes me think that the protein didn't even transfer over in the first place. The post-doc in my lab says even if you can't see the protein bands at the lower sizes on the membrane doesn't mean it's not there. So what I tried to do now, which probably won't work, is re-probe my membrane paper with actin antibody to see if the actin would show up at least. Then I'll stain with ponceau to see if the actin shows up on the membrane and then develop on film. This way I'll know whether the problem is with my antibodies itself or perhaps its the ECL or film development problem. But my membrane has been dried out on the cassette for about a day so I don't think the re-probing will work but worth a try?!
It should still work. I've done that before. Let me know what you find!
It still works and the actin antibody worked as well although the signals were very weak. So now at least I know the mistake is somewhere in the antibodies or the transfer itself and not in the solutions. But this time, I can see the outlines of the membrane paper on the film. I still don't understand why I wouldn't see the outline before even if the other antibodies didn't work. Of course, I am still plagued by the fact that most of my proteins remained on my gel eventhough the ladder completely transferred over and even went through my membrane.
I'm sure your antibody works, but some antibodies need more antigen than others depending on its purity and specificity. The fact that all your protein didn't transfer over could be due to a limit of your membrane in binding capacity? The amount of ladder protein was probably substantially lower than that of the other lanes, and if it were higher some of it could have stayed behind. Just a guess. Do you make your own gels? Maybe it has something to do with the lower gel buffer you use? I've read that soaking your gel in transfer buffer before putting it in the sandwich helps, but I have not tested it.
I do soak my gels before transferring. If the amount of ladder is lower than that of my proteins, then wouldn't more of the proteins be able to transfer over on the membrane instead of staying on the gel? I made my own gel at 12% but what could be wrong with the lower tris buffer? That makes me a little concerned since I had run into some trouble with that. I had to add a whooping 50 ml worth of HCl in order to get it to the right pH which bothered me but since my gels ran fine I didn't think much of it.
as far as the buffer, if your recipe specified tris-HCl and you made it using tris-base, I would expect that more HCl would be required to obtain the correct pH. this might result in a molarity that is not correct. you may choose to remake the buffer, checking that all components are the right form
The recipe actually called for Tris base but indicated to adjust the pH to 8.8 (for lower buffer) or 6.8 (for upper buffer) with HCl. Even so, that is an abnormally high volume of HCl to use for pH adjustment I'd say. If my proteins ran fine on my gel, there has to be a way to transfer it over to the membrane without running the whole thing over?!
Do you have SDS in your gel buffer?
I use: 0.4% SDS, 236.4g/L Tris-HCl, pH = 8.8
I read that SDS will help proteins out of the gel, and MeOH in your transfer buffer will help them bind to the membrane. I use 20% MeOH. What is the pH of your transfer buffer?
Here are some troubleshooting pointers I pulled from my Bio-Rad guide for poor transfer:
1. Proteins near their isoelectric point at the pH of the buffer will transfer poorly. Try a more basic or aidic transfer buffer to increase protein mobility.
2. Filter paper too dry
3. Gel percentage too high, specifically the Bis (crosslinker) percentage.
4. Try 0.2uM nitrocellulose
The pH of my transfer buffer is around 9. I do add SDS to my sds gel and to my transfer buffer as well. I am running it again this time at a higher voltage or perhaps longer time to see if that will make a difference. But I am growing pretty weary. I'm thinking could it be my sample buffer as well since the only difference between my ladder and proteins samples I could think of is the sample buffer I added to the protein samples. What recipe do you use for sample buffer? O btw, I am posting this issue under a new topic since we are no longer discussing antibodies anymore, if you don't mind you can post replies under the new topic as well =)