Use this calculator to help you correctly dilute your antibodies for western blotting. To correctly dilute secondary antibodies for western blotting it is recommended to first make an antibody working stock solution of 1 µg/mL and dilute further for the final antibody incubation solution. Recommended dilutions for use with Thermo Scientific ECL substrates and Alexa Fluor Plus conjugates are below.
Primary Antibody Dilution
Select desired final volume for antibody incubation
Selection desired final antibody dilution
Selection desired final antibody dilution
Selection desired final antibody dilution
Selection desired final antibody dilution
Final concentration
1:100
µL antibody stock
100
µL of buffer to create 10 mL of final antibody solution
9,900
Final concentration
1:250
µL antibody stock
40
µL of buffer to create 10 mL of final antibody solution
9,960
Final concentration
1:500
µL antibody stock
20
µL of buffer to create 10 mL of final antibody solution
9,980
Final concentration
1:1,000
µL antibody stock
10
µL of buffer to create 10 mL of final antibody solution
9,990
Final concentration
1:2,000
µL antibody stock
5
µL of buffer to create 10 mL of final antibody solution
9,995
Final concentration
1:2,500
µL antibody stock
4
µL of buffer to create 10 mL of final antibody solution
9,996
Final concentration
1:3,000
µL antibody stock
3.3
µL of buffer to create 10 mL of final antibody solution
9,997
Final concentration
1:5,000
µL antibody stock
2
µL of buffer to create 10 mL of final antibody solution
9,998
Final concentration
1:10,000
µL antibody stock
1
µL of buffer to create 10 mL of final antibody solution
9,999
Final concentration
1:15,000
µL antibody stock
Volume too low, increase to 15 mL final volume
µL of buffer to create 10 mL of final antibody solution
-
Final concentration
1:20,000
µL antibody stock
Volume too low, increase to 20 mL final volume
µL of buffer to create 10 mL of final antibody solution
-
Final concentration
1:100
µL antibody stock
35
µL of buffer to create 3.5 mL of final antibody solution
3,465
Final concentration
1:250
µL antibody stock
14
µL of buffer to create 3.5 mL of final antibody solution
3,486
Final concentration
1:500
µL antibody stock
7
µL of buffer to create 3.5 mL of final antibody solution
3,493
Final concentration
1:1,000
µL antibody stock
3.5
µL of buffer to create 3.5 mL of final antibody solution
3,497
Final concentration
1:2,000
µL antibody stock
1.75
µL of buffer to create 3.5 mL of final antibody solution
3,498
Final concentration
1:2,500
µL antibody stock
1.4
µL of buffer to create 3.5 mL of final antibody solution
3,499
Final concentration
1:3,000
µL antibody stock
1.17
µL of buffer to create 3.5 mL of final antibody solution
3,499
Final concentration
1:5,000
µL antibody stock
0.7
µL of buffer to create 3.5 mL of final antibody solution
3,499
Final concentration
1:10,000
µL antibody stock
Volume too low, increase to 10 mL final volume
µL of buffer to create 3.5 mL of final antibody solution
Final concentration
1:15,000
µL antibody stock
Volume too low, increase to 15 mL final volume
µL of buffer to create 3.5 mL of final antibody solution
-
Final concentration
1:20,000
µL antibody stock
Volume too low, increase to 20 mL final volume
µL of buffer to create 3.5 mL of final antibody solution
-
Final concentration
1:100
µL antibody stock
150
µL of buffer to create 15 mL of final antibody solution
14,850
Final concentration
1:250
µL antibody stock
60
µL of buffer to create 15 mL of final antibody solution
14,940
Final concentration
1:500
µL antibody stock
30
µL of buffer to create 15 mL of final antibody solution
14,970
Final concentration
1:1,000
µL antibody stock
15
µL of buffer to create 15 mL of final antibody solution
14,985
Final concentration
1:2,000
µL antibody stock
7,5
µL of buffer to create 15 mL of final antibody solution
14,992
Final concentration
1:2,500
µL antibody stock
6
µL of buffer to create 15 mL of final antibody solution
14,994
Final concentration
1:3,000
µL antibody stock
5
µL of buffer to create 15 mL of final antibody solution
14,995
Final concentration
1:5,000
µL antibody stock
3
µL of buffer to create 15 mL of final antibody solution
14,997
Final concentration
1:10,000
µL antibody stock
1,5
µL of buffer to create 15 mL of final antibody solution
14,998
Final concentration
1:15,000
µL antibody stock
1
µL of buffer to create 15 mL of final antibody solution
14,999
Final concentration
1:20,000
µL antibody stock
Volume too low, increase to 20 mL final volume
µL of buffer to create 15 mL of final antibody solution
-
Final concentration
1:100
µL antibody stock
200
µL of buffer to create 20 mL of final antibody solution
19,800
Final concentration
1:250
µL antibody stock
80
µL of buffer to create 20 mL of final antibody solution
19,920
Final concentration
1:500
µL antibody stock
40
µL of buffer to create 20 mL of final antibody solution
19,960
Final concentration
1:1,000
µL antibody stock
20
µL of buffer to create 20 mL of final antibody solution
19,980
Final concentration
1:2,000
µL antibody stock
10
µL of buffer to create 20 mL of final antibody solution
19,990
Final concentration
1:2,500
µL antibody stock
8
µL of buffer to create 20 mL of final antibody solution
19,992
Final concentration
1:3,000
µL antibody stock
6,7
µL of buffer to create 20 mL of final antibody solution
19,993.3
Final concentration
1:5,000
µL antibody stock
4
µL of buffer to create 20 mL of final antibody solution
19,996
Final concentration
1:10,000
µL antibody stock
2
µL of buffer to create 20 mL of final antibody solution
19,998
Final concentration
1:15,000
µL antibody stock
1.3
µL of buffer to create 20 mL of final antibody solution
19,998.7
Final concentration
1:20,000
µL antibody stock
1
µL of buffer to create 20 mL of final antibody solution
19,999
Secondary Antibody Dilution
Step 1: Preparing 1 µg/mL antibody working stock solution
Select antibody starting stock concentration
Volume of antibody working stock solution required
Add volume below of antibody starting stock
To volume below of buffer diluent
20 mL
6.7 µL
19,993.3 µL
15 mL
5 µL
14,995 µL
10 mL
3.3 µL
9,996.7 µL
2 mL
0.7 µL
1,999.3 µL
Volume of antibody working stock solution required
Add volume below of antibody starting stock
To volume below of buffer diluent
20 mL
8 µL
19,992 µL
15 mL
6 µL
14,994 µL
10 mL
4 µL
9,996 µL
2 mL
0.8 µL
1,999.2 µL
Volume of antibody working stock solution required
Add volume below of antibody starting stock
To volume below of buffer diluent
20 mL
10 µL
19,990 µL
15 mL
7.5 µL
14,992.5 µL
10 mL
5 µL
9,995 µL
2 mL
1 µL
1,999 µL
1 mL
0.5 µL
999.5 µL
Volume of antibody working stock solution required
Add volume below of antibody starting stock
To volume below of buffer diluent
20 mL
20 µL
19,990 µL
15 mL
15 µL
14,985 µL
10 mL
10 µL
9,990 µL
2 mL
2 µL
1,998 µL
1 mL
1 µL
999 µL
0.5 mL
0.5 µL
499.5 µL
Volume of antibody working stock solution required
Add volume below of antibody starting stock
To volume below of buffer diluent
20 mL
40 µL
19,960 µL
15 mL
30 µL
14,970 µL
10 mL
20 µL
9,980 µL
2 mL
4 µL
1,996 µL
1 mL
2 µL
998 µL
0.5 mL
1 µL
499 µL
Volume of antibody working stock solution required
Add volume below of antibody starting stock
To volume below of buffer diluent
20 mL
100 µL
19,800 µL
15 mL
75 µL
14,925 µL
10 mL
50 µL
9,950 µL
2 mL
10 µL
1,990 µL
1 mL
5 µL
995 µL
0.5 mL
2.5 µL
497.5 µL
Volume of antibody working stock solution required
Add volume below of antibody starting stock
To volume below of buffer diluent
20 mL
200 µL
19,800 µL
15 mL
150 µL
14,850 µL
10 mL
100 µL
9,900 µL
2 mL
20 µL
1,980 µL
1 mL
10 µL
990 µL
0.5 mL
5 µL
495 µL
Volume of antibody working stock solution required
Add volume below of antibody starting stock
To volume below of buffer diluent
20 mL
400 µL
19,600 µL
15 mL
300 µL
14,985 µL
10 mL
200 µL
9,800 µL
2 mL
40 µL
1,960 µL
1 mL
20 µL
980 µL
0.5 mL
10 µL
490 µL
Step 2: Prepare final antibody dilution
Select desired final volume for antibody incubation
Selection desired final antibody dilution
Selection desired final antibody dilution
Selection desired final antibody dilution
Selection desired final antibody dilution
Final concentration
1:1,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
No further dilution required
µL of 1 µg/mL antibody working stock
10,000
µL of buffer to create 10 mL of final antibody solution
0
µg/ml
1.000
ng/ml
1,000
Final concentration
1:2,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:2
µL of 1 µg/mL antibody working stock
5,000
µL of buffer to create 10 mL of final antibody solution
5,000
µg/ml
0.5
ng/ml
500
Final concentration
1:3,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:3
µL of 1 µg/mL antibody working stock
3,333
µL of buffer to create 10 mL of final antibody solution
6,667
µg/ml
0.333
ng/ml
333
Final concentration
1:5,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:5
µL of 1 µg/mL antibody working stock
2,000
µL of buffer to create 10 mL of final antibody solution
8,000
µg/ml
0.200
ng/ml
200
Final concentration
1:10,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:10
µL of 1 µg/mL antibody working stock
1,000
µL of buffer to create 10 mL of final antibody solution
9,000
µg/ml
0.100
ng/ml
100
Final concentration
1:15,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:15
µL of 1 µg/mL antibody working stock
667
µL of buffer to create 10 mL of final antibody solution
9,333
µg/ml
0.067
ng/ml
67
Final concentration
1:20,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:20
µL of 1 µg/mL antibody working stock
500
µL of buffer to create 10 mL of final antibody solution
9,500
µg/ml
0.050
ng/ml
50
Final concentration
1:25,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:25
µL of 1 µg/mL antibody working stock
400
µL of buffer to create 10 mL of final antibody solution
9,600
µg/ml
0.040
ng/ml
40
Final concentration
1:50,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:50
µL of 1 µg/mL antibody working stock
200
µL of buffer to create 10 mL of final antibody solution
9,800
µg/ml
0.020
ng/ml
20
Final concentration
1:75,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:75
µL of 1 µg/mL antibody working stock
133
µL of buffer to create 10 mL of final antibody solution
9,867
µg/ml
0.013
ng/ml
13
Final concentration
1:100,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:100
µL of 1 µg/mL antibody working stock
133
µL of buffer to create 10 mL of final antibody solution
9,900
µg/ml
0.010
ng/ml
10
Final concentration
1:125,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:125
µL of 1 µg/mL antibody working stock
80
µL of buffer to create 10 mL of final antibody solution
9,920
µg/ml
0.008
ng/ml
8
Final concentration
1:150,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:150
µL of 1 µg/mL antibody working stock
67
µL of buffer to create 10 mL of final antibody solution
9,933
µg/ml
0.007
ng/ml
7
Final concentration
1:200,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:200
µL of 1 µg/mL antibody working stock
50
µL of buffer to create 10 mL of final antibody solution
9,950
µg/ml
0.005
ng/ml
5
Final concentration
1:250,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:250
µL of 1 µg/mL antibody working stock
40
µL of buffer to create 10 mL of final antibody solution
9,960
µg/ml
0.004
ng/ml
4
Final concentration
1:500,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:500
µL of 1 µg/mL antibody working stock
20
µL of buffer to create 10 mL of final antibody solution
9,980
µg/ml
0.002
ng/ml
2
Final concentration
1:1,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
0
µL of 1 µg/mL antibody working stock
3,500
µL of buffer to create 3.5 mL of final antibody solution
-
µg/ml
1.000
ng/ml
1,000
Final concentration
1:5,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:2
µL of 1 µg/mL antibody working stock
1,750
µL of buffer to create 3.5 mL of final antibody solution
1,750
µg/ml
0.5
ng/ml
500
Final concentration
1:5,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:3
µL of 1 µg/mL antibody working stock
1,167
µL of buffer to create 3.5 mL of final antibody solution
2,333
µg/ml
0.333
ng/ml
333
Final concentration
1:5,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:5
µL of 1 µg/mL antibody working stock
875
µL of buffer to create 3.5 mL of final antibody solution
2,625
µg/ml
0.200
ng/ml
200
Final concentration
1:10,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:10
µL of 1 µg/mL antibody working stock
350
µL of buffer to create 3.5 mL of final antibody solution
3,150
µg/ml
0.100
ng/ml
100
Final concentration
1:15,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:15
µL of 1 µg/mL antibody working stock
233
µL of buffer to create 3.5 mL of final antibody solution
3,267
µg/ml
0.067
ng/ml
67
Final concentration
1:20,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:20
µL of 1 µg/mL antibody working stock
175
µL of buffer to create 3.5 mL of final antibody solution
3,325
µg/ml
0.050
ng/ml
50
Final concentration
1:25,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:25
µL of 1 µg/mL antibody working stock
140
µL of buffer to create 3.5 mL of final antibody solution
3,360
µg/ml
0.040
ng/ml
40
Final concentration
1:50,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:50
µL of 1 µg/mL antibody working stock
70
µL of buffer to create 3.5 mL of final antibody solution
3,430
µg/ml
0.020
ng/ml
20
Final concentration
1:75,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:75
µL of 1 µg/mL antibody working stock
47
µL of buffer to create 3.5 mL of final antibody solution
3,453
µg/ml
0.013
ng/ml
13
Final concentration
1:100,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:100
µL of 1 µg/mL antibody working stock
35
µL of buffer to create 3.5 mL of final antibody solution
3,465
µg/ml
0.010
ng/ml
10
Final concentration
1:125,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:125
µL of 1 µg/mL antibody working stock
28
µL of buffer to create 3.5 mL of final antibody solution
3,472
µg/ml
0.008
ng/ml
8
Final concentration
1:150,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:150
µL of 1 µg/mL antibody working stock
23
µL of buffer to create 3.5 mL of final antibody solution
3,477
µg/ml
0.007
ng/ml
7
Final concentration
1:200,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:200
µL of 1 µg/mL antibody working stock
17.5
µL of buffer to create 3.5 mL of final antibody solution
3,482.5
µg/ml
0.005
ng/ml
5
Final concentration
1:250,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:250
µL of 1 µg/mL antibody working stock
14
µL of buffer to create 3.5 mL of final antibody solution
3,486
µg/ml
0.004
ng/ml
4
Final concentration
1:500,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:500
µL of 1 µg/mL antibody working stock
7
µL of buffer to create 3.5 mL of final antibody solution
3,493
µg/ml
0.002
ng/ml
2
Final concentration
1:1,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
No further dilution required
µL of 1 µg/mL antibody working stock
15,000
µL of buffer to create 15 mL of final antibody solution
0
µg/ml
1.000
ng/ml
1,000
Final concentration
1:2,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:2
µL of 1 µg/mL antibody working stock
7.5000
µL of buffer to create 15 mL of final antibody solution
7.5000
µg/ml
0.5
ng/ml
500
Final concentration
1:3,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:3
µL of 1 µg/mL antibody working stock
5,000
µL of buffer to create 15 mL of final antibody solution
10,000
µg/ml
0.333
ng/ml
333
Final concentration
1:5,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:5
µL of 1 µg/mL antibody working stock
3,000
µL of buffer to create 15 mL of final antibody solution
12,000
µg/ml
0.200
ng/ml
200
Final concentration
1:10,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:10
µL of 1 µg/mL antibody working stock
1,500
µL of buffer to create 15 mL of final antibody solution
13,500
µg/ml
0.100
ng/ml
100
Final concentration
1:15,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:15
µL of 1 µg/mL antibody working stock
1,000
µL of buffer to create 15 mL of final antibody solution
14,000
µg/ml
0.067
ng/ml
67
Final concentration
1:20,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:20
µL of 1 µg/mL antibody working stock
750
µL of buffer to create 15 mL of final antibody solution
14,250
µg/ml
0.05
ng/ml
50
Final concentration
1:25,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:25
µL of 1 µg/mL antibody working stock
600
µL of buffer to create 15 mL of final antibody solution
14,400
µg/ml
0.040
ng/ml
40
Final concentration
1:50,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:50
µL of 1 µg/mL antibody working stock
300
µL of buffer to create 15 mL of final antibody solution
14,700
µg/ml
0.020
ng/ml
20
Final concentration
1:75,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:75
µL of 1 µg/mL antibody working stock
200
µL of buffer to create 15 mL of final antibody solution
14,800
µg/ml
0.013
ng/ml
13
Final concentration
1:100,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:100
µL of 1 µg/mL antibody working stock
150
µL of buffer to create 15 mL of final antibody solution
14,850
µg/ml
0.010
ng/ml
10
Final concentration
1:125,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:125
µL of 1 µg/mL antibody working stock
120
µL of buffer to create 15 mL of final antibody solution
14,880
µg/ml
0.008
ng/ml
8
Final concentration
1:150,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:150
µL of 1 µg/mL antibody working stock
100
µL of buffer to create 15 mL of final antibody solution
14,900
µg/ml
0.007
ng/ml
7
Final concentration
1:200,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:200
µL of 1 µg/mL antibody working stock
75
µL of buffer to create 15 mL of final antibody solution
14,925
µg/ml
0.005
ng/ml
5
Final concentration
1:250,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:250
µL of 1 µg/mL antibody working stock
60
µL of buffer to create 15 mL of final antibody solution
14,940
µg/ml
0.004
ng/ml
4
Final concentration
1:500,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:500
µL of 1 µg/mL antibody working stock
30
µL of buffer to create 15 mL of final antibody solution
14,970
µg/ml
0.002
ng/ml
2
Final concentration
1:1,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
No further dilution required
µL of 1 µg/mL antibody working stock
20,000
µL of buffer to create 20 mL of final antibody solution
0
µg/ml
1.000
ng/ml
1,000
Final concentration
1:2,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:2
µL of 1 µg/mL antibody working stock
10,000
µL of buffer to create 20 mL of final antibody solution
10,000
µg/ml
0.5
ng/ml
500
Final concentration
1:3,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:3
µL of 1 µg/mL antibody working stock
6,667
µL of buffer to create 20 mL of final antibody solution
13,333
µg/ml
0.333
ng/ml
333
Final concentration
1:5,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:5
µL of 1 µg/mL antibody working stock
4,000
µL of buffer to create 20 mL of final antibody solution
16,000
µg/ml
0.2
ng/ml
200
Final concentration
1:10,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:10
µL of 1 µg/mL antibody working stock
2,000
µL of buffer to create 20 mL of final antibody solution
18,000
µg/ml
0.1
ng/ml
100
Final concentration
1:15,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:15
µL of 1 µg/mL antibody working stock
1,333
µL of buffer to create 20 mL of final antibody solution
18,667
µg/ml
0.067
ng/ml
67
Final concentration
1:20,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:20
µL of 1 µg/mL antibody working stock
1,000
µL of buffer to create 20 mL of final antibody solution
19,000
µg/ml
0.05
ng/ml
50
Final concentration
1:25,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:25
µL of 1 µg/mL antibody working stock
800
µL of buffer to create 20 mL of final antibody solution
19,200
µg/ml
0.04
ng/ml
40
Final concentration
1:50,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:50
µL of 1 µg/mL antibody working stock
400
µL of buffer to create 20 mL of final antibody solution
19,600
µg/ml
0.02
ng/ml
20
Final concentration
1:75,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:75
µL of 1 µg/mL antibody working stock
267
µL of buffer to create 20 mL of final antibody solution
19,733
µg/ml
0.013
ng/ml
13
Final concentration
1:100,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:100
µL of 1 µg/mL antibody working stock
200
µL of buffer to create 20 mL of final antibody solution
19,800
µg/ml
0.01
ng/ml
10
Final concentration
1:125,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:125
µL of 1 µg/mL antibody working stock
160
µL of buffer to create 20 mL of final antibody solution
19,840
µg/ml
0.008
ng/ml
8
Final concentration
1:150,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:150
µL of 1 µg/mL antibody working stock
133
µL of buffer to create 20 mL of final antibody solution
19,867
µg/ml
0.007
ng/ml
7
Final concentration
1:200,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:200
µL of 1 µg/mL antibody working stock
100
µL of buffer to create 20 mL of final antibody solution
19,900
µg/ml
0.005
ng/ml
5
Final concentration
1:250,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:250
µL of 1 µg/mL antibody working stock
80
µL of buffer to create 20 mL of final antibody solution
19,920
µg/ml
0.004
ng/ml
4
Final concentration
1:500,000
Second dilution factor after preparing 1 µg/mL antibody stock solution
1:500
µL of 1 µg/mL antibody working stock
40
µL of buffer to create 20 mL of final antibody solution
A dilution factor of X:Y means you need to add X μL of a stock solution to (Y − X) μL of diluent, where Y is the final volume you require (in μL). For example, to prepare a solution with a dilution of 1:250 from an antibody stock, add 1 volume of the stock solution to 249 volumes of the diluent. To prepare 10 mL (or 10,000 μL) of a 1:250 dilution from an antibody stock, add (10,000 μL / 250) = 40 μL of antibody stock, to (10,000 μL diluent − 40 μL antibody stock) = 9,960 μL of diluent.
Performing your own antibody dilution calculations
The equation below is used determine the volume of the starting stock required to prepare the standardized 1 μg/mL working stock: CsVs = CfVf
Cs is the starting antibody stock concentration (which is a known value) Vs is the volume of starting antibody stock required (value to be determined) Cf is the final antibody concentration desired (which is 1 μg/mL for the standardized working stock) Vf is the final volume of antibody solution desired (you may need to prepare 1 mL, 2 mL, or 10 mL, depending on the final dilution required)
This equation will need to be solved for Vs: Vs = CfVf / Cs
Note: The concentration variables must use the same units (e.g., mg/mL or μg/mL), and the volume variable must use the same units (e.g., mL or μL) in order to perform this calculation appropriately. Unit conversion may be required.
