I am a beginner of western blot and my target protein if 124kDA whereas the endogeneous controls are 41kDA and 55kDA. I did wet transfer at 100V, 80mins and successfully got the band for target protein.But both of my endogeneous controls did not show any band. I thought that it might be due to the overshoot of low MW proteins. Next day I cut the gel and did transfer at 100V for 40mins for the smaller ones but this time also there was no band.But in every cases ladder was perfectly transferred in the membrane.Should I reduce the time? Can I transfer the target and endogeneous control at same duration and same voltage?
Since all the bands of your ladder were transfered nicely, I would doubt that your low MW proteins have migrated through the membrane.
Usually it's the other way round (the 50 kDa loading control is fine and the 15 kDa target protein is not). Could there be anything wrong with the two control antibodies (gone bad, too old, precipitated, bound to the tube etc)? Have you ever successfully used them yourself? Did you use the correct corresponding secondary antibody/ies?
Did you perform a staining of the membrane after blotting (eg with Ponceau) to get an idea how well the overall transfer went?
@ Ali & Robert
Thank you so much.Is there any chance that the proteins were swimmed out of the blot but the ladder sticked to the membrane?
What is the difference between your "target" and your "endogenous controls"? If all else is just fine (fresh antibody, the correct secondary antibody(s) etc.), it could be that the reason you are not detecting the "endogenous controls" is because you simply have not loaded enought of your lysate to detect them. Try loading a range of protein amounts on your gel - 10ug, 30ug and 50ug.
@Mirsada
The difference is 83kDA. I have already loaded 10ug and 20ug but in none of them, I did detect my protein.
As stated above make sure your antibodies are working, this also includes the secondary. There can be a lot of variability in antibody detection (does anyone else in the lab use these antibodies?). If these are endogenous proteins then they may well be in very small amounts and therefore difficult to detect especially if your antibodies are not that sensitive. As suggested load more protein >30 microg
You could play around with the conditions of WB (One condition does not always suite all antibodies): antibody concentration, buffer used: TBS or PBS with Tween 20, blocker used (over blocking can lead to loss of detection).
Other suggestions are to change your control protein if all else is not working.
Hello Maynak,
as told above, 41 and 55 kDa are no low molecular weights for western blotting. If you see the acurate molecular weight marker, then it is assumed that your blotting procedure was working. You wrote, that you want detect endogenous control proteins in your sample. Are you sure, that your antibody is able to detect these endogenous proteins? Sometimes, there are differences depending if you use reduced or non reduced samples. To be sure, that you procedure is working you can stain one blot with coomassie blue. What are the control proteins? Do you strip the membrane before detecting the controls?
One more suggestion concerning the endogenous control primary antibodies (since we are all guessing here):
As some people have asked already: Have these antibodies succesfully been used in a Western Blot or maybe so far only for other applications? Some antibodies work fine just for one technique (WB, Immunofluorescence, ELISA etc.). A nice control antibody for staining PFA-fixed cells may not yield any signal by WB and vice versa...
no bands can arise due to many reasons related to antibody, antigen, or buffer used. If an improper antibody is used, either primary or secondary, the band will not show. In addition, the concentration of the antibody should be appropriate as well; if the concentration is too low, the signal may not be visible. It is important to remember that some antibodies are not to be used for western blot. Another reason for no visible bands is the lowest concentration or absence of the antigen. In this case, antigen from another source can be used to confirm whether the problem lies with the sample or with other elements, such as the antibody. Moreover, prolonged washing can also decrease the signal. Buffers can also contribute to the problem. It should be ensured that buffers like the transfer buffer, TBST, running buffer and ECL are all new and noncontaminated. If the buffers are contaminated with sodium azide, it can inactivate HRP.. Similarly, weak signals can be caused by low concentration of antibody or antigen. Increasing exposure time can also help to make the band clearer. Another reason could be nonfat dry milk masking the antigen. In this case use BSA or decrease the amount of milk used
Good luck
You already got excellent suggestions. Since your protein of interest is of a higher molecular weight (>100), I would suggest that you could use a lower percentage gel instead of the usual 4%-10% combination. Perhaps you could try a 7% resolving gel, the resolution is better and the bands separate out nicely. As for your endogenous controls, I would suggest you go back to the literature and see the dilutions of the antibodies they used. Also sometimes, you have different isoforms of the endogenous controls in different systems. For example, a beta actin will not be suitable control for all the cells or tissue systems. So, you could look into that as well. However, quantifying the expression of your protein of interest with respect to the total protein is considered to be the ideal now.
All the best.
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