Synthesis of EGF


Protein Assays

Assay #1

September 1, 2016
BioBasics Better BCA Kit

BSA Standards Prep

  1. Label tubes and prepare sample on ice
  2. Pipetted desired volume of Solution D outlined in Table 1.
  3. Added desired volume of BSA standard outlined in Table 1 and pipetted solution up and down to mix.
  4. Stored on ice while preparing samples and solutions.

Table 1: BSA Protein Standards

Tube Volume of BSA Standard (uL) Solution D Volume (uL) Final Concentration (ug/mL)
1 120 ul of 2 mg/mL std 120 1000
2 150 ul of above 50 750
3 100 ul of above 50 500
4 80 ul of above 80 250
5 80 ul of above 80 125
6 60 ul of above 40 75
7 20 ul of above 40 25
8 0 80 0


Solution AB Prep

  1. Make 1x Solution A using volumetric ration of 4:1 (water to concentrated solution A). Pipetted 4 mL of MilliQ water and added 1 mL of concentrated solution A into a 15 mL Falcon tube. Some of the solution looked precipitated/cloudy. Solution cleared after addition of Solution B in next steps.
  2. Add 200 uL of solution B to 15 mL Falcon tube containing 5 mL of 1x Solution A to form Solution AB. Vortex for 5 seconds to mix and store on ice.
  3. I ended up making too little of Solution AB. Made another 5 mL Solution A with 100 uL Solution B separately and added to later wells (will explain below).
  4. (8 standards + 3 samples * 3 dilutions) * 2 duplicates * 200 uL into each well = 6800 uL or 6.8 mL

Solution CD Prep

  1. Add 250 uL of MilliQ water to 25 uL of Solution D into a microfuge tube.
  2. In a separate microfuge tube, weighted ~ 10mg of Solution C. Actual Weight added = ~ 10.5-11.0 mg.
  3. Transferred diluted solution D into tube containing weighed out Solution C. Vortex for 1 minute to dissolve.
  4. Store on ice.

Sample Prep

  1. Sample prepared according to Table 2,3, and 4 at varying dilutions (1x, 2x dilute, 10x dilute)
  2. 6 uL (~9%) of 10 wt% SDS added to Sample #2 and Sample #3 to disassociate the liposomes. BCA assay tolerance to SDS is below 5% so by adding 6 uL, final concentration of SDS ~0.9% so shouldn’t effect absorbance signal.
  3. Tubes mixed by vortexing 5 sec.

Table 2: Preparation of EGF Sample (Sample #1)

Tube Volume of Sample (uL) Solution D Volume (uL) Sample Dilution
1-1 60 0 1x
1-2 30 30 2x dilute
1-3 6 64 10x dilute

Table 3: Preparation of Before Dialysis Liposome Sample (Sample #2)

Tube Volume of Sample (uL) Solution D Volume (uL) SDS Volume (uL) Sample Dilution
2-1 60 0 6 1x (60/66)
2-2 30 30 6 2x dilute (30/66)
2-3 6 54 6 10x dilute (6/66)

Table 4: Preparation of Final Liposome Sample (Sample #3)

Tube Volume of Sample (uL) Solution D Volume (uL) SDS Volume (uL) Sample Dilution
2-1 60 0 6 1x (60/66)
2-2 30 30 6 2x dilute (30/66)
2-3 6 54 6 10x dilute (6/66)

BCA Assay/Plate Loading

  1. Used clear flat bottom 96 well plate for assay.
  2. Added 5 uL of standards/sample according to Table 5. Add directly to center of well.
  3. Added 5 uL of solution CD and incubate a 37°C for 30 minutes (12:16 am –> 12:46 pm). Note: 10 uL of std/sample with solution CD does not cover bottom of the well. Solution CD pipetted into middle of well into std/sample solution. Plate gently shaken by hand (back and forth and in circles). Samples containing SDS filled bottom of well due to lower surface tension. Bubbles formed when pipetting into SDS samples (tried to minimize). Covered with aluminum foil during incubation.
  4. Add 200 uL of Solution AB into standards and samples. Only prepared 5 mL of Solution AB initially so ran out at after Well B8. Wells containing 2nd batch of Solution AB = B9, C8, C9, A11, A12, B11, B12, C11, C12.
  5. Gently mixed by shaking back and forth and in circles. Incubated with Solution AB at 37°C for another 30 mins, covered with foil (12:55 am –> 1:25 pm)
  6. Red plate 3 times. Twice using standard settings (100 ms read time) and last one at 1:41 pm using 200 ms read time. Absorbance at 562 nm.

Table 5: 96 Well Plate Configuration

  1 2 3 4 5 6 7 8 9 10 11 12
A 1000 1000     1-1 1-1   2-1 2-1   3-1 3-1
B 750 750     1-2 1-2   2-2 2-2   3-2 3-2
C 500 500     1-3 1-3   2-3 2-3   3-3 3-3
D 250 250                    
E 125 125                    
F 75 75                    
G 25 25                    
H 0 0                    

columns 1 and 2 are standards

Assay #2

September 13th, 2016
Micro BCA Protein Assay Kit from Hallam Lab
Present: Nathan, Edward, Valerie, Chanpreet

Preparation of BSA Protein Standards

Table 1: BSA Standard Solutions Preparation

Vial Standard Solution (uL) Buffer (uL) Concentration (ug/mL)
A 100 900 200
B 200 of A 800 40
C 500 of B 500 20
D 500 of C 500 10
E 500 of D 500 5
F 500 of D 500 2.5
G 400 of E 600 1
H 500 of H 500 0.5
I 0 500 0

Preparation of Working Solution

(# of standards + # of samples) * (# of replicates) * (volume required) = total volume of working solution

(9 standards + (2 samples * 2 dilutions)) * 2 replicates * 150 uL volume = 3.9 mL of working solution required –> will prepare ~ 5 mL of working solution

Preparation of Samples

Table 2: Sample Preparation

Tube Stock Sample (uL) Buffer (uL) Final Concentration
E2 250 250 2x dil
E10 50 450 10x dil
F2 250 250 2x dil
F10 50 450 10x dil

Loading the Plate

Table 3: 96 Well Plate Configuration

A o o x x o o x o o x o o
B o o x x o o x o o x o o
C o o x x o o x o o x o o
D o o x x x x x x x x x x
E o o x x x x x x E10 E2 F10 F2
F o o x x x x x x E10 E2 F10 F2
G o o x a b c d e f g h i
H o o x a b c d e f g h i

a-i: standards
E10, E2, F10, F2: samples
x: empty
o: previously filled

Data


  Adj Abs Protein (ug/mL) Adjusted Protein (ug/mL)  
EGF 1:10 0.166428 20.35698 203.5698 687.0482
EGF 1:2 0.378051 47.88852 95.77704 323.2475
Lipo 1:10 0.407811 51.76021 517.6021  
Lipo 1:2 1.405749 181.589 363.1781  
Slope 0.007687      
Intercept -0.00995      

Thiolation of EGF

Maleimide/Thiol Assay