Good day
I was told a few weeks back that GAPDH cannot be used as a loading control in differentiated cells since its expression does not stay constant. Has anybody heard anything about this?
the best thing to do would be to use another one or two loading controls initially along with gapdh and compare your results. when you find a loading control that is providing consistent results, use that for the remainder of the experiments. for example, we used 18s with gapdh in our differentiation experiments, and both showed similar results.
Ideally, you should use more than one control, especially if you want to publish your result in high impact journal. GAPDH, BETA Actin, or S18 is examples of stable controls.
Never heard about this issue. What cell type you are talking about? Possible. If possible you may want to check it out.
There are several options for Western blotting (we are talking about Westerns and not qRT-PCR??) to make sure you loaded the same amount of protein and can normalize for differences. As a simple loading control you can make sure you really did a good job with determining the protein concentration, perhaps remeasure; quickly stain the blot with Ponceau S and evaluate whether there is indeed very similar loading before you continue with antibody exposure.
If you want to have an additional layer for normalization, it makes perfect sense to use 2 or 3 such normalizers. GAPDH is often a choice but tubulin, actin, histone H3 are "alternatives" that you should use if you are in doubt that the normalizer of your choice is actually not evenly expressed in you samples despite even loading. Technically loading control and normalizer are not the same but most of the time fulfill the same function: to make sure that you can evaluate whether your protein of interest is differentially expressed in your experimental samples versus control samples.
I use GAPDH as a loading control in HEK293T cells. But this may not be the case across cells from different tissues. I hope you find a suitable loading control.
GAPDH, tubulin, total actin (not beta-actin only) are good set for normalization. You have to check and compare in your conditions. Loading control is preliminar, but it is better to use several housekeeping proteins.
Hi Toni
If the problem is about Real-Time PCR I recommend as suggest colleges using several different controls and then test them using programs such as GeNorm or Bestkeeper. Good luck
Hi Toni.
This is indeed a controversial topic. Many people will say that you can use and many others will say that you can't use. Indeed, many people think that the cell expresses the same genes throughout the day, for instance; however, it is known that the cells expresses several enzymes and protein in determined hours.
Thus, you should first make some experiments to verify if theses markers (use more than one) are ''constitutive'', i.e. if they do not vary significantly during your experiments. I don't like using GAPDH because it is a metabolic enzyme - thus it can vary depending on the cellular context. B-actin is a structural protein that can vary as well.
If you are using q-PCR assay you can choose 18s-RNA and others loading control. If you are performing WB; then you can try B-actin and GAPDH.
Again, it is very controversial and you should test more than one control in order to find the best control for your model of study.
I hope it helps.
Thank you all for the replies. I received an article from the company we bought the antibody from so I'll go by that. I will be sure to use more than one loading control in future, but for now I am finished in the lab.
- Badges
- Science method