can we use Comassie blue for loading control after the immunoblotting and imaging of the primary of interest on membrane? Is it reversible?
Dear Priyamvada,
I would not use comassie blue for staining immunoblot membranes. We use the non-covalent protein stain "Ponceau S" (0.1% Ponceau S in 5% CH3COOH).
For best results, remove the membrane from the blotting transfer chamber. Wash membrane with distilled water to remove the methanol and add the stain for 10 min. The stain can be reused so don't throw in away after use. Remove stain and wash membrane in distilled water. This will show you how equal your loading is. Now, to remove the stain wash in TSBT (TBS with 0.1%Tween20).
As I said, this gives the best results, you should still be able to look at your loading after blocking and antibody exposure, although you will have much more "membrane" staining. Also, keep in mind that the CH3COOH in the staining solution might also cause your antibody to be removed from its target, as antibody binding is dependent on pH and ionic strength of the buffer.
I hope this helps.
Greetings
Wynand
Dear Priyamvada,
I would not use comassie blue for staining immunoblot membranes. We use the non-covalent protein stain "Ponceau S" (0.1% Ponceau S in 5% CH3COOH).
For best results, remove the membrane from the blotting transfer chamber. Wash membrane with distilled water to remove the methanol and add the stain for 10 min. The stain can be reused so don't throw in away after use. Remove stain and wash membrane in distilled water. This will show you how equal your loading is. Now, to remove the stain wash in TSBT (TBS with 0.1%Tween20).
As I said, this gives the best results, you should still be able to look at your loading after blocking and antibody exposure, although you will have much more "membrane" staining. Also, keep in mind that the CH3COOH in the staining solution might also cause your antibody to be removed from its target, as antibody binding is dependent on pH and ionic strength of the buffer.
I hope this helps.
Greetings
Wynand
I agree with Wynand. We typically stain with Ponceau S before blocking. In our experience, it does not affect immuno-staining. Comassie will impact antibody-antigen interactions, and is best performed in the gel, not the membrane. Ponceau S is definitely the easier route.
I agree with Wynand and Chris, use Ponceau S stain. A great way to look at the transfer of your gel without affecting downstream immunoblotting. Also, there is Amido Black stain too.
I agree with everything that has been said above but maybe I misinterpretated your question.
Ponceua, as metioned will serve as a transfer control since it's not very quantitative. If you are interested in a loading control (to show you have the same sample amount throughout the lanes) you must strip your membrane and then use a second antibody that targets proteins which expression doesn't change due to your biological question (i.e cell activation, tissue specificity, cell maturation, etc).
You can use:
- alpha actin, GAPDH, beta tubulin (for WCL);
- HSP60 (for mithocondria);
- laminin, PCNA, hystone H3 (for nuclear);
- NaK ATPase (for membrane),;
- alpha actin, beta tubulin (for cytoskeleton);
- transferrin (for serum).
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