Following

Following

Hi, Leong Chee Yang

As mentioned, you have loaded 28ul of your plant protein extract (concentration- 0.7032ug/ul). It corresponds to ~19.7ug of protein which is a sufficient amount to be visible after staining. Stain the gel well with fresh coomassie blue for 20mins and then destain throughly. As you have loaded protein extract, you will get a smear of protein bands instead of a single band (obtained for purified protein).

Mr Tushar has recommended fresh staining with coomassive blue, try to extend the de-staining time. You can consider a more sensitive protocol for staining and destaining. Combination of imidazole-zinc reverse staining and Coomassie Blue-based fast staining method has proven to be increase sensitivity significantly

check this journal:

doi: 10.1371/journal.pone.0081696

• RuBPS staining is more sensitive than CBB, and quite cheap if you make your own reagent (doi:10.1007/978-1-49395-87452-08\_11 has detailed instructions).
• At the border between stacking and separating gel I notice a band of presumably aggregated material. Heat the protein in sample buffer with SDS and DTT (better than ßME due to more appropriate redox potential, doi:10.1021/bi00892a002) to 60 °C for 15 min, do not boil.
• While the bands of standard proteins are crisp, the bands in your sample appear smeared. This can be the result of too much salt, a sample pH very different from neutral or nucleic acids. Perhaps you should precipitate the protein and then dissolve it directly in 1 x sample buffer (doi:10.1016/0003-2697(84)90782-6).
• Protein assays measure the absorbance relative to that of a standard protein. Since different proteins differ in their colour yield with different assays, depending on the combination of reference protein and assay method chosen, the results for a given sample may vary by a factor of 20 (sic!). Thus, perhaps you need to concentrate your protein and load a little more. Concentration is easily done by the precipitation method mentioned above.
• Your stacking gels appear rather small, add at least 5 mm so that protein bands can be focussed properly. See doi:10.1111/j.1749-6632.1964.tb14207.x for the theoretical background.
• For a minigel, use 10 mA constant current during stacking and 20 mA during separation. If your electrophoresis chamber has loops for water cooling, use them.
• During sample preparation, ensure that you have protease inhibitors present.

Frank Abimbola Ogundolie

May I know can I do imidazole-zinc reverse staining before in gel trypsination?

Does any effect on my protein bands?

Engelbert Buxbaum

May I proceed to the next steps (In gel trypsination) after doing negative staining followed by CBS and destain with 4mM EDTA?

Does any effect on my protein bands for carrying out in gel trypsination ?

The advantage of Zn/imidazol-staining is that it is fully reversible. If you cut out the (un)stained band, extract any Zn with EDTA and then wash out the EDTA with your digestion buffer for a few minutes, digestion should work fine.

Hi

I fully agree with Engelbert. On the top of both concentrating and sepparating gel remains proteins. During electrophoresis, this proteins (precipitated,denatured or with HMW) may be solubilzed and running giving smear. I suggest you a centrifugation step before applying sample in gel 10000 g, 5min. On the other hand plant extracts are rich in phenols, aldehydes, ketones and other that disturbs the electrophoretic profile. Don't worry about it, this typical of this kind of samples

Regards, Oscar

Puede intentar tinción con nitrato de plata para una mejor detección en la segunda imagen, y revisar el protocolo de extracción para la primera imagen o aumentar la cantidad de muestra ajustando el protocolo.

You can try silver nitrate staining for better detection for the second image, and change the extraction protocol for the first image or increase the amount of sample by adjusting the protocol.