I'm fairly new to Western Blotting but was first taught to dilute my antibodies in my blocking buffer (which is 5% NDM in TBST). Recently, during some troubleshooting, I was advised to get out of the habit of always using TBST (more specifically just using my 5%NDM in TBST) to dilute antibodies because sometimes the antibodies are not compatible with the Tween-20 and need to be in TBS (again blocking in TBS rather than TBST) instead. The company data sheets for the antibodies in question (Santa Cruz Biotech) don't specify one way or the other so I'm wondering if there's another way to tell (TBS or TBST for an antibody dilution) or if anyone has any recommendations (and rationale) for one way over the other.
It could depend on the type of your membrane: Nitrocellulose vs. PVDF
Ioana, I have not used PBS. The ECL kit we use recommends TBS/TBST for most of the steps so for simplicity, we use TBS/TBST for everything.
Salamah, PVDF.
As you mentioned Santa Cruz Antibodies, most of these Abs are not species specific, they can bind a protein from mouse, rat, pig, goat, etc. whatever you have diluted ur Abs in TBS/T, or 5% milk in PBS + 0.1% tween 20 (as I usually do), it is not easily to get one specific band of the target protein, Try Cell Signaling company Although it is more expensive!
Good Luck
Sorry, I've missunderstood your question. Actually, I am diluting my antibodies into TBST 5% milk, but some people in my lab are using Odyssey Blocking Buffer from LI-COR. We don't use ECL, and in stead we scan our blots with Odyssay scan, after useing fluorescent secondary antibodies. I personnaly don't recomand Santa Crouz either for antibodies (I had problems with alot of antibodies from them, and they recomand to use at high concentration such as 1/200). I actually by antibodies either from Sigma (the HPA antibodies are highly specific), or I buy from Cell Signaling or Ambcam. I hope I hepled with this comment!
I do not use Tween unless I have too. There is an extensive literature which shows that it alters the results seen in Western blots and ELISAs. Thus, I only use it as a last resort. I dilute Abs in TBS or PBS containing 0.2-0.5% casein. I have found this to be the best protein to add to antibody solutions.
The main purpose of Tween in antibody solutions is to prevent non-specific binding. In the case of a very weak primary antibody, or a very weakly expressed protein, I would omit it. Otherwise, I use it routinely in WB primary and secondary solutions. Of course, if the manufacturer recommends against it, you should take that into consideration. But I haven't seen any such cases.
You can use 5% milk in TBST as mentioned earlier in posts. It works better and then wash your membrane with 1% TBST for 10X 3 times. It works for almost all the Ab's I personally used todate. The Tween 20 is used in 5% milk in TBST just to avoid non specific binding of your Ab with proteins. If some of the Ab's still bound,the 1% TBST can remove it during washing steps. Wish you all the best
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology
- Antibodies