Protein quant

If in sample is in urea lysis buffer, use Bradford assay (5X) dilute in water—use multichannel pipette


 * Make 500uL more than necessary
 * Add 3uL ULB to standards! (13uL total…10uL standard + 3uL buffer)

If any other buffer—use pierce BCA kit

BCA Kit set up
Quant buffer: 50 parts clear buffer : 1 part blue buffer, use 200uL buffer per sample

Use standard curve, 20uL each of :


 * 1000ng/uL (20ug)


 * 500ng/uL (10ug)


 * 250ng/uL (5ug)


 * 125ng/uL (2.5ug)

Load the 20uL of each standard into wells of flat 96well plate
 * water (0ug)

Load 3uL each sample

Add 200uL per well of BCA mix (50:1), mix via pipetting

Wait ~10minutes (will see color develop)

How to use plate reader
Go to plate reader with plate and usb key

Turn on in back, plug in usb

Put plate into machine w/o lid (A1 in upper left)

Softmax pro app


 * Cmd—new
 * Settings à read at 560nm
 * Templateàpick wellsà newà unknownsàok (Makes column of readout)
 * Read
 * Fileà export


 * Quit, take plate


 * Shut drawer (hit “drawer”)


 * Turn off in back


 * eject usb

Protein Quant Calculation

 * Open txt file from excel


 * subtract background from every read first (so blank = 0)


 * make an xy scatter plot (y value = read, x value = protein content)


 * add linear trendline and display equation


 * can get rid of the most nonlinear sample

we have the y value (softmax pro reading) for our unknown samples, now we just have to solve for x. This gives us the total protein content of that well. Now, we divide by 3uL for protein sample concentration.

Super concentrated samples: dilution series
1:5 and 1:15 (overall)

3uL sample + 12uL empty buffer = 1:5 dilution

3uL of 1:5 + 6uL empty buffer = 1:15 dilution overall (1:3 dilution of 1:5 material)

Load 3uL of 1:5 dilution and 1uL of 1:15 dilution for quant