If you are careful enough and do not allow the bromophenol blue tracking dye to elute out of the gel during electrophoresis, 7.5% gel will work.

Since you are new in WB, I would suggest you to go with 10% SDS-PAGE to resolve 20-60kDa protein.

The Laemmli sample buffer is usually prepared as 5X stock and accordingly diluted in the final loading mix with protein to 1X containing 5% beta-ME (for reducing condition).

thanks Dr Amritlal Mandal for your suggestion.. it is possible to keep the diluted laemmli buffer with sample and uses it again and again or it must be prepared freshly to be loaded into sds-page gel?

What size gel will you run (large vs mini)? If you will run large gels (that separate overnight), I would recommend that you use 10 or 12% SDS-PAGE. If you run small gels, 10% would also be good. Will you cast your own gel or do you purchase pre-casted gels? What type of transfer apparatus will you use? You will need to be careful in your transfer to prevent over-transferring your 20kDa protein in order to get sufficient transfer of your 60kDa protein. For loading (Laemmli) buffer, I use a 4x concentration with the appropriate amount of BME added for reducing conditions.

You can use 12% of acrylamide gel for 20-60kDa proteins. protein loading buffer you can use 6x concentration.

Dear Khairul,

I suggest you to make 5X Laemmli sample buffer (LSB) stock with no beta-ME added. Prepare your samples for electrophoresis by adding required volume of 5X LSB to be diluted finally to 1X, water and beta -ME (5% of final volume).

If you are preparing samples for 50 ul well volume then follow the following protocol

Final vol: 50 ul

5X LSB: 10 ul

Protein added: x ul (depends on concentration and required amount you want to load)

water: [50-(10+x+2.5)] ul

beta-ME: 2.5 ul (5% of 50 ul)

Mix the sample thoroughly by vortexing and heat at 95deg C in water bath heater for 5 min, briefly centrifuge to settle down the loading mix at the bottom of the microfuge tube and load onto the gel.

For short term use you may keep 5X LSB at RT and save the rest of the stock at 4 deg C. Warm up the stock 5X LSB in a water bath set at 50 deg C to dissolve SDS present in the buffer as SDS settles down at RT or at lower temp which creates problem in pipetting.

For membrane proteins heating the samples is not recommended. Use 37-40 deg C lower temp bath for longer time (30-60 min) to reduce the proteins. Heating at 95 deg C cause protein aggregation and the proteins in the gel run like smear with either very high background or low and diffused band intensities.

For non reducing conditions you have to take different strategies with modified protocol for sample preparations.

Hope that helps.

If you use small gel then 7.5% may give you problem since it never separate out anything below 25K for me. But 7.5% gel may work for big gel. Use PVDF or 0.22 nitrocellulose for transfer. 2X, 5X, or 6X LSB make no difference to me and it depends on the protein content in the cell lysates.

Thanks for all suggestions. I really appreciated it. I will try to use some of your suggestion and see if it's working for me.

4-20% Gradient for almost anything. If you don't pour your own. Great for publications! Nice tight bands.