6/14/21

Dear Sushil,

I will try to answer your questions.

1. If you need to run the PAGE @ 4C, you can run the separation in a cold room. If you don't have a cold room, you can try inserting PAGE apparatus in a refrigerator. The power supply should be kept outside. Note that during the separation, some heat is generated, so the temperature in the apparatus will probably not be @ 4C; however, that may not be a big problem.

2. It's important to know the starting concentration of your protein solution. Be sure to calculate the new concentration after dilution w/ sample buffer. Both the Bradford and Lowry assay methods are OK; the Bradford is more sensitive. You can also use the BCA (Bi-Cinchoninic Acid) method. This is also very good. It is similar to the Lowry, but is more sensitive. The procedure for the BCA assay can be found by doing a google search.

The amount of protein you apply to each lane of the gel will depend to some extent on the protein stain you are using. If you use Coomassie Blue, which is very sensitive, you can detect just a few uGrams of protein per lane. You can always try applying increasing amounts of protein to adjacent lanes until you find an amount of protein that you can easily visualize

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3. If you are trying to detect peroxidase (POD) using an activity stain, heating the sample should not be done, as it will inactivate the enzyme. Heating is usually done with SDS-PAGE. This is conducted under denaturing conditions, usually w/ mercapto-ethanol to dissociate enzymes w/ multiple subunits, so heating (100C) is done.

4. There are a # of protein stains for use w/ gels. One of the most popular is Coomassie Brilliant Blue (CBB). After the PAGE is done, the gel is immersed in CBB for ~1 hr, then de-stained in acetic acid/MeOH to remove background dye from the gel. The procedures and recipes can be found doing a google search.

For an activity stain, you can use guiacol. This functions as a proton donor to allow POD to convert H2O2 to 2 molecules of H2O . You can also use ABTS (2,2′-Azinobis-(3-Ethylbenzothiazoline-6-Sulfonic Acid). This produces a dark bluish-green color with POD. I think (?) it is more sensitive than guiacol.

One thing you could do is apply you duplicate samples to multiple wells in your slab gel. After PAGE, cut the gel in half vertically. Stain one half of the slab w/ CBB and the other half w/ H2O2 + guiacol (or ABTS). Then you can line up the CBB bands w/ the activity stain bands.

I hope this information helps you.

Bill Colonna Ames, Iowa, USA [email protected]

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Zagazig University, Egypt. [email protected]

2. The Soret absorbance at 403 nm to measure the absorbance of HRP is more reliable and accurate compared to absorbance at 280 nm (ε403 = 1.02 × 105 M−1 cm−1 as the molar absorption coefficient)

Hi Sushil, in addition to performing all the above-mentioned steps, one thing which might help is to extract the enzyme by column chromatography itself followed by the native PAGE instead of using a cell lysate(if that is what you use). Additionally, the biological activity of peroxidase is to neutralise H202, so it might undergo conformational change and rapidly use substrate leading to reduced efficiency in staining. You could try to use higher H202 concentrations.