Hi,

If you can measure the total protein of the 500 flies in 1ml , you can calculate the total protein content of 1 fly.

Depending on your staining you can find if that is enough for SDS-PAGE.

You can adjust the amount of RIPA buffer to an usable amount.

with kind regards,

Ronald

Thank you Mr. Ronald!

So, you are advising me to use 2 µl RIPA buffer for one fly. Tough job but I will try and let you know.

Thanks again.

You can easily degrade the tissue of the organism with a homogenizer or sonication and extract the proteins.

Hi Pijush,

I meant to say that if one fly gives enough protein for SDS-PAGE, you can adjust the amount of ripa buffer (say up to 20µl). It will lower the concentration of protein, but it will be usable for lysis and SDS-PAGE .

WIth kind regards,

Ronald

Hi Ronald,

I got your message. Now it seems quite easier than I thought. I am not hopeful in getting a good result from one fly because protein yield will be too low (< 3–5 µg/µl). However, I will go through BSA method after extracting from a healthy whitefly. If possible then I will proceed to the next.

Thank you very much.

Sincere regards,

Pijush

Hi Pijush,

3-5µg might be enough to get result when you use a silverstaining for you gel.

With kind regards,

Ronald

Hi,

Don't bother with RIPA buffer if you want to extract as much protein as possible from the fly for a gel.

Add 12 ul of SDS-PAGE sample buffer directly to the fly, heat at 85C for 10 min. Let cool. Spin for 5 minutes at 10K on a microfuge to pellet insoluble material, then transfer 10 ul to the gel directly. The SDS in the sample buffer will extract just about all the protein from a sample that small without a lot of mechanical disruption. I predict you will have enough protein to see a nice band pattern with silver stain.

Hi,

Mary Cristine.

I liked your answer.

Thank you

Great! Did it work, i hope?