We use both of these protocol and both works fine in our hands. In protocol A, at step 4, we do centrifuge at 15000g for 10-15 min (4C) to get most of the cells in pellet.

Hi Iwan, 

I prefer the  B protocol, I prefer the B protocol, it is similar to what I use and have obtained a good performance.  I have obtained a yield of 2-3 ug /ul protein followed the protocol below:

1) Remove a medium from a dish plate (66mm), with approximately 80-90% of the confluence.

2) then make two washes with 1 ml PBS  1X

3) Remove all the PBS, because it can leave the protein lysate more diluted than expected

4) Then, add 50 uL of the Lysis Buffer (of you choice), scrape the cells on a box with ice. Then, transfer the contents into microtubes and hold 1 hour at 4º  for complete cell dissociation. During the transfer, make a vigorous homogenization of content with micopipette. 

5) After 1 hour,  centrifuge the microtubes at 13.000 rpm, for 15 minutes. 

6) Remove 2 ul of sobrenadant and performed the protein quantification. 

Hope this information might help!

We use both of these protocol and both works fine in our hands. In protocol A, at step 4, we do centrifuge at 15000g for 10-15 min (4C) to get most of the cells in pellet.

I think both protocols are equally good, but I personally use a variation of your protocol A. I scrape the plates with two quantities of icPBS to catch any stray cells before spinning down at 1500 for 4 minutes. I also rock my lysates at 4C for 30 minutes to ensure the cells have lysed.

I've recently added sonication to my protocol before spinning out debris, which has quadrupled by protein yield. However, I was only vortexing to homogenize my samples previously, not pipetting. 

I would normally go for your protocol B but usually if I am using smaller well surface, e.g for up to 24-well plate. If you are using anything bigger than that, like your 60mm dish, then the 50ul of lysis buffer is too little to cover the surface and by the time you scratch all the cells, your lysis buffer will be simply all bubbles. So for bigger wells or plates I would go for the protocol A. 

I would go with A because with the larger volume, you will be less likely to get all of your protein.  50ul is a very small volume and just missing 1 or 2 ul, you are losing a lot of protein.  I also sonicate just to make sure everything is dissociated.

If you have to pick one, I'd go with option B, as I prefer to scrape cells in the presence of lysis buffer to inhibit any enzyme activity that could destroy your proteins upon cell lysis (physical scraping of cells will induce some cell lysis).  However, I've always had better protein yield if I instead trypsinize, spin cells down, aspirate, wash with 1mL of PBS, re-spin then aspirate again and freeze on dry ice and store at -80C.  I regularly get protein quantities from 2-12ug/uL or more depending on the volume of lysis buffer I add to the pellet.  The other nice thing about doing it this way is if you have multiple conditions or time points, you can harvest at different time points or different days and still lyse at the same time, decreasing your likelihood of different lysis conditions and uneven loading.

i have used the second method with petit variants, so i would recomend it has the better, anyway you could try both of them if it is posible and compare results with your cels.