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 concentration1:100
µL antibody stock100
µL of buffer to create 10 mL of final antibody solution9,900
Final concentration1:250
µL antibody stock40
µL of buffer to create 10 mL of final antibody solution9,960
Final concentration1:500
µL antibody stock20
µL of buffer to create 10 mL of final antibody solution9,980
Final concentration1:1,000
µL antibody stock10
µL of buffer to create 10 mL of final antibody solution9,990
Final concentration1:2,000
µL antibody stock5
µL of buffer to create 10 mL of final antibody solution9,995
Final concentration1:2,500
µL antibody stock4
µL of buffer to create 10 mL of final antibody solution9,996
Final concentration1:3,000
µL antibody stock3.3
µL of buffer to create 10 mL of final antibody solution9,997
Final concentration1:5,000
µL antibody stock2
µL of buffer to create 10 mL of final antibody solution9,998
Final concentration1:10,000
µL antibody stock1
µL of buffer to create 10 mL of final antibody solution9,999
Final concentration1:15,000
µL antibody stockVolume too low, increase to 15 mL final volume
µL of buffer to create 10 mL of final antibody solution-
Final concentration1:20,000
µL antibody stockVolume too low, increase to 20 mL final volume
µL of buffer to create 10 mL of final antibody solution-
Final concentration1:100
µL antibody stock35
µL of buffer to create 3.5 mL of final antibody solution3,465
Final concentration1:250
µL antibody stock14
µL of buffer to create 3.5 mL of final antibody solution3,486
Final concentration1:500
µL antibody stock7
µL of buffer to create 3.5 mL of final antibody solution3,493
Final concentration1:1,000
µL antibody stock3.5
µL of buffer to create 3.5 mL of final antibody solution3,497
Final concentration1:2,000
µL antibody stock1.75
µL of buffer to create 3.5 mL of final antibody solution3,498
Final concentration1:2,500
µL antibody stock1.4
µL of buffer to create 3.5 mL of final antibody solution3,499
Final concentration1:3,000
µL antibody stock1.17
µL of buffer to create 3.5 mL of final antibody solution3,499
Final concentration1:5,000
µL antibody stock0.7
µL of buffer to create 3.5 mL of final antibody solution3,499
Final concentration1:10,000
µL antibody stockVolume too low, increase to 10 mL final volume
µL of buffer to create 3.5 mL of final antibody solution
Final concentration1:15,000
µL antibody stockVolume too low, increase to 15 mL final volume
µL of buffer to create 3.5 mL of final antibody solution-
Final concentration1:20,000
µL antibody stockVolume too low, increase to 20 mL final volume
µL of buffer to create 3.5 mL of final antibody solution-
Final concentration1:100
µL antibody stock150
µL of buffer to create 15 mL of final antibody solution14,850
Final concentration1:250
µL antibody stock60
µL of buffer to create 15 mL of final antibody solution14,940
Final concentration1:500
µL antibody stock30
µL of buffer to create 15 mL of final antibody solution14,970
Final concentration1:1,000
µL antibody stock15
µL of buffer to create 15 mL of final antibody solution14,985
Final concentration1:2,000
µL antibody stock7,5
µL of buffer to create 15 mL of final antibody solution14,992
Final concentration1:2,500
µL antibody stock6
µL of buffer to create 15 mL of final antibody solution14,994
Final concentration1:3,000
µL antibody stock5
µL of buffer to create 15 mL of final antibody solution14,995
Final concentration1:5,000
µL antibody stock3
µL of buffer to create 15 mL of final antibody solution14,997
Final concentration1:10,000
µL antibody stock1,5
µL of buffer to create 15 mL of final antibody solution14,998
Final concentration1:15,000
µL antibody stock1
µL of buffer to create 15 mL of final antibody solution14,999
Final concentration1:20,000
µL antibody stockVolume too low, increase to 20 mL final volume
µL of buffer to create 15 mL of final antibody solution-
Final concentration1:100
µL antibody stock200
µL of buffer to create 20 mL of final antibody solution19,800
Final concentration1:250
µL antibody stock80
µL of buffer to create 20 mL of final antibody solution19,920
Final concentration1:500
µL antibody stock40
µL of buffer to create 20 mL of final antibody solution19,960
Final concentration1:1,000
µL antibody stock20
µL of buffer to create 20 mL of final antibody solution19,980
Final concentration1:2,000
µL antibody stock10
µL of buffer to create 20 mL of final antibody solution19,990
Final concentration1:2,500
µL antibody stock8
µL of buffer to create 20 mL of final antibody solution19,992
Final concentration1:3,000
µL antibody stock6,7
µL of buffer to create 20 mL of final antibody solution19,993.3
Final concentration1:5,000
µL antibody stock4
µL of buffer to create 20 mL of final antibody solution19,996
Final concentration1:10,000
µL antibody stock2
µL of buffer to create 20 mL of final antibody solution19,998
Final concentration1:15,000
µL antibody stock1.3
µL of buffer to create 20 mL of final antibody solution19,998.7
Final concentration1:20,000
µL antibody stock1
µL of buffer to create 20 mL of final antibody solution19,999

Secondary Antibody Dilution

Step 1: Preparing 1 µg/mL antibody working stock solution

Select antibody starting stock concentration

Volume of antibody working stock solution requiredAdd volume below of antibody starting stockTo volume below of buffer diluent
20 mL6.7 µL19,993.3 µL
15 mL5 µL14,995 µL
10 mL3.3 µL9,996.7 µL
2 mL0.7 µL1,999.3 µL
Volume of antibody working stock solution requiredAdd volume below of antibody starting stockTo volume below of buffer diluent
20 mL8 µL19,992 µL
15 mL6 µL14,994 µL
10 mL4 µL9,996 µL
2 mL0.8 µL1,999.2 µL
Volume of antibody working stock solution requiredAdd volume below of antibody starting stockTo volume below of buffer diluent
20 mL10 µL19,990 µL
15 mL7.5 µL14,992.5 µL
10 mL5 µL9,995 µL
2 mL1 µL1,999 µL
1 mL0.5 µL999.5 µL
Volume of antibody working stock solution requiredAdd volume below of antibody starting stockTo volume below of buffer diluent
20 mL20 µL19,990 µL
15 mL15 µL14,985 µL
10 mL10 µL9,990 µL
2 mL2 µL1,998 µL
1 mL1 µL999 µL
0.5 mL0.5 µL499.5 µL
Volume of antibody working stock solution requiredAdd volume below of antibody starting stockTo volume below of buffer diluent
20 mL40 µL19,960 µL
15 mL30 µL14,970 µL
10 mL20 µL9,980 µL
2 mL4 µL1,996 µL
1 mL2 µL998 µL
0.5 mL1 µL499 µL
Volume of antibody working stock solution requiredAdd volume below of antibody starting stockTo volume below of buffer diluent
20 mL100 µL19,800 µL
15 mL75 µL14,925 µL
10 mL50 µL9,950 µL
2 mL10 µL1,990 µL
1 mL5 µL995 µL
0.5 mL2.5 µL497.5 µL
Volume of antibody working stock solution requiredAdd volume below of antibody starting stockTo volume below of buffer diluent
20 mL200 µL19,800 µL
15 mL150 µL14,850 µL
10 mL100 µL9,900 µL
2 mL20 µL1,980 µL
1 mL10 µL990 µL
0.5 mL5 µL495 µL
Volume of antibody working stock solution requiredAdd volume below of antibody starting stockTo volume below of buffer diluent
20 mL400 µL19,600 µL
15 mL300 µL14,985 µL
10 mL200 µL9,800 µL
2 mL40 µL1,960 µL
1 mL20 µL980 µL
0.5 mL10 µL490 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 concentration1:1,000
Second dilution factor after preparing 1 µg/mL antibody stock solutionNo further dilution required
µL of 1 µg/mL antibody working stock10,000
µL of buffer to create 10 mL of final antibody solution0
µg/ml1.000
ng/ml1,000
Final concentration1:2,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:2
µL of 1 µg/mL antibody working stock5,000
µL of buffer to create 10 mL of final antibody solution5,000
µg/ml0.5
ng/ml500
Final concentration1:3,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:3
µL of 1 µg/mL antibody working stock3,333
µL of buffer to create 10 mL of final antibody solution6,667
µg/ml0.333
ng/ml333
Final concentration1:5,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:5
µL of 1 µg/mL antibody working stock2,000
µL of buffer to create 10 mL of final antibody solution8,000
µg/ml0.200
ng/ml200
Final concentration1:10,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:10
µL of 1 µg/mL antibody working stock1,000
µL of buffer to create 10 mL of final antibody solution9,000
µg/ml0.100
ng/ml100
Final concentration1:15,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:15
µL of 1 µg/mL antibody working stock667
µL of buffer to create 10 mL of final antibody solution9,333
µg/ml0.067
ng/ml67
Final concentration1:20,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:20
µL of 1 µg/mL antibody working stock500
µL of buffer to create 10 mL of final antibody solution9,500
µg/ml0.050
ng/ml50
Final concentration1:25,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:25
µL of 1 µg/mL antibody working stock400
µL of buffer to create 10 mL of final antibody solution9,600
µg/ml0.040
ng/ml40
Final concentration1:50,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:50
µL of 1 µg/mL antibody working stock200
µL of buffer to create 10 mL of final antibody solution9,800
µg/ml0.020
ng/ml20
Final concentration1:75,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:75
µL of 1 µg/mL antibody working stock133
µL of buffer to create 10 mL of final antibody solution9,867
µg/ml0.013
ng/ml13
Final concentration1:100,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:100
µL of 1 µg/mL antibody working stock133
µL of buffer to create 10 mL of final antibody solution9,900
µg/ml0.010
ng/ml10
Final concentration1:125,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:125
µL of 1 µg/mL antibody working stock80
µL of buffer to create 10 mL of final antibody solution9,920
µg/ml0.008
ng/ml8
Final concentration1:150,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:150
µL of 1 µg/mL antibody working stock67
µL of buffer to create 10 mL of final antibody solution9,933
µg/ml0.007
ng/ml7
Final concentration1:200,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:200
µL of 1 µg/mL antibody working stock50
µL of buffer to create 10 mL of final antibody solution9,950
µg/ml0.005
ng/ml5
Final concentration1:250,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:250
µL of 1 µg/mL antibody working stock40
µL of buffer to create 10 mL of final antibody solution9,960
µg/ml0.004
ng/ml4
Final concentration1:500,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:500
µL of 1 µg/mL antibody working stock20
µL of buffer to create 10 mL of final antibody solution9,980
µg/ml0.002
ng/ml2
Final concentration1:1,000
Second dilution factor after preparing 1 µg/mL antibody stock solution0
µL of 1 µg/mL antibody working stock3,500
µL of buffer to create 3.5 mL of final antibody solution-
µg/ml1.000
ng/ml1,000
Final concentration1:5,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:2
µL of 1 µg/mL antibody working stock1,750
µL of buffer to create 3.5 mL of final antibody solution1,750
µg/ml0.5
ng/ml500
Final concentration1:5,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:3
µL of 1 µg/mL antibody working stock1,167
µL of buffer to create 3.5 mL of final antibody solution2,333
µg/ml0.333
ng/ml333
Final concentration1:5,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:5
µL of 1 µg/mL antibody working stock875
µL of buffer to create 3.5 mL of final antibody solution2,625
µg/ml0.200
ng/ml200
Final concentration1:10,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:10
µL of 1 µg/mL antibody working stock350
µL of buffer to create 3.5 mL of final antibody solution3,150
µg/ml0.100
ng/ml100
Final concentration1:15,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:15
µL of 1 µg/mL antibody working stock233
µL of buffer to create 3.5 mL of final antibody solution3,267
µg/ml0.067
ng/ml67
Final concentration1:20,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:20
µL of 1 µg/mL antibody working stock175
µL of buffer to create 3.5 mL of final antibody solution3,325
µg/ml0.050
ng/ml50
Final concentration1:25,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:25
µL of 1 µg/mL antibody working stock140
µL of buffer to create 3.5 mL of final antibody solution3,360
µg/ml0.040
ng/ml40
Final concentration1:50,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:50
µL of 1 µg/mL antibody working stock70
µL of buffer to create 3.5 mL of final antibody solution3,430
µg/ml0.020
ng/ml20
Final concentration1:75,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:75
µL of 1 µg/mL antibody working stock47
µL of buffer to create 3.5 mL of final antibody solution3,453
µg/ml0.013
ng/ml13
Final concentration1:100,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:100
µL of 1 µg/mL antibody working stock35
µL of buffer to create 3.5 mL of final antibody solution3,465
µg/ml0.010
ng/ml10
Final concentration1:125,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:125
µL of 1 µg/mL antibody working stock28
µL of buffer to create 3.5 mL of final antibody solution3,472
µg/ml0.008
ng/ml8
Final concentration1:150,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:150
µL of 1 µg/mL antibody working stock23
µL of buffer to create 3.5 mL of final antibody solution3,477
µg/ml0.007
ng/ml7
Final concentration1:200,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:200
µL of 1 µg/mL antibody working stock17.5
µL of buffer to create 3.5 mL of final antibody solution3,482.5
µg/ml0.005
ng/ml5
Final concentration1:250,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:250
µL of 1 µg/mL antibody working stock14
µL of buffer to create 3.5 mL of final antibody solution3,486
µg/ml0.004
ng/ml4
Final concentration1:500,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:500
µL of 1 µg/mL antibody working stock7
µL of buffer to create 3.5 mL of final antibody solution3,493
µg/ml0.002
ng/ml2
Final concentration1:1,000
Second dilution factor after preparing 1 µg/mL antibody stock solutionNo further dilution required
µL of 1 µg/mL antibody working stock15,000
µL of buffer to create 15 mL of final antibody solution0
µg/ml1.000
ng/ml1,000
Final concentration1:2,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:2
µL of 1 µg/mL antibody working stock7.5000
µL of buffer to create 15 mL of final antibody solution7.5000
µg/ml0.5
ng/ml500
Final concentration1:3,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:3
µL of 1 µg/mL antibody working stock5,000
µL of buffer to create 15 mL of final antibody solution10,000
µg/ml0.333
ng/ml333
Final concentration1:5,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:5
µL of 1 µg/mL antibody working stock3,000
µL of buffer to create 15 mL of final antibody solution12,000
µg/ml0.200
ng/ml200
Final concentration1:10,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:10
µL of 1 µg/mL antibody working stock1,500
µL of buffer to create 15 mL of final antibody solution13,500
µg/ml0.100
ng/ml100
Final concentration1:15,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:15
µL of 1 µg/mL antibody working stock1,000
µL of buffer to create 15 mL of final antibody solution14,000
µg/ml0.067
ng/ml67
Final concentration1:20,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:20
µL of 1 µg/mL antibody working stock750
µL of buffer to create 15 mL of final antibody solution14,250
µg/ml0.05
ng/ml50
Final concentration1:25,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:25
µL of 1 µg/mL antibody working stock600
µL of buffer to create 15 mL of final antibody solution14,400
µg/ml0.040
ng/ml40
Final concentration1:50,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:50
µL of 1 µg/mL antibody working stock300
µL of buffer to create 15 mL of final antibody solution14,700
µg/ml0.020
ng/ml20
Final concentration1:75,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:75
µL of 1 µg/mL antibody working stock200
µL of buffer to create 15 mL of final antibody solution14,800
µg/ml0.013
ng/ml13
Final concentration1:100,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:100
µL of 1 µg/mL antibody working stock150
µL of buffer to create 15 mL of final antibody solution14,850
µg/ml0.010
ng/ml10
Final concentration1:125,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:125
µL of 1 µg/mL antibody working stock120
µL of buffer to create 15 mL of final antibody solution14,880
µg/ml0.008
ng/ml8
Final concentration1:150,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:150
µL of 1 µg/mL antibody working stock100
µL of buffer to create 15 mL of final antibody solution14,900
µg/ml0.007
ng/ml7
Final concentration1:200,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:200
µL of 1 µg/mL antibody working stock75
µL of buffer to create 15 mL of final antibody solution14,925
µg/ml0.005
ng/ml5
Final concentration1:250,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:250
µL of 1 µg/mL antibody working stock60
µL of buffer to create 15 mL of final antibody solution14,940
µg/ml0.004
ng/ml4
Final concentration1:500,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:500
µL of 1 µg/mL antibody working stock30
µL of buffer to create 15 mL of final antibody solution14,970
µg/ml0.002
ng/ml2
Final concentration1:1,000
Second dilution factor after preparing 1 µg/mL antibody stock solutionNo further dilution required
µL of 1 µg/mL antibody working stock20,000
µL of buffer to create 20 mL of final antibody solution0
µg/ml1.000
ng/ml1,000
Final concentration1:2,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:2
µL of 1 µg/mL antibody working stock10,000
µL of buffer to create 20 mL of final antibody solution10,000
µg/ml0.5
ng/ml500
Final concentration1:3,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:3
µL of 1 µg/mL antibody working stock6,667
µL of buffer to create 20 mL of final antibody solution13,333
µg/ml0.333
ng/ml333
Final concentration1:5,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:5
µL of 1 µg/mL antibody working stock4,000
µL of buffer to create 20 mL of final antibody solution16,000
µg/ml0.2
ng/ml200
Final concentration1:10,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:10
µL of 1 µg/mL antibody working stock2,000
µL of buffer to create 20 mL of final antibody solution18,000
µg/ml0.1
ng/ml100
Final concentration1:15,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:15
µL of 1 µg/mL antibody working stock1,333
µL of buffer to create 20 mL of final antibody solution18,667
µg/ml0.067
ng/ml67
Final concentration1:20,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:20
µL of 1 µg/mL antibody working stock1,000
µL of buffer to create 20 mL of final antibody solution19,000
µg/ml0.05
ng/ml50
Final concentration1:25,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:25
µL of 1 µg/mL antibody working stock800
µL of buffer to create 20 mL of final antibody solution19,200
µg/ml0.04
ng/ml40
Final concentration1:50,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:50
µL of 1 µg/mL antibody working stock400
µL of buffer to create 20 mL of final antibody solution19,600
µg/ml0.02
ng/ml20
Final concentration1:75,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:75
µL of 1 µg/mL antibody working stock267
µL of buffer to create 20 mL of final antibody solution19,733
µg/ml0.013
ng/ml13
Final concentration1:100,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:100
µL of 1 µg/mL antibody working stock200
µL of buffer to create 20 mL of final antibody solution19,800
µg/ml0.01
ng/ml10
Final concentration1:125,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:125
µL of 1 µg/mL antibody working stock160
µL of buffer to create 20 mL of final antibody solution19,840
µg/ml0.008
ng/ml8
Final concentration1:150,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:150
µL of 1 µg/mL antibody working stock133
µL of buffer to create 20 mL of final antibody solution19,867
µg/ml0.007
ng/ml7
Final concentration1:200,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:200
µL of 1 µg/mL antibody working stock100
µL of buffer to create 20 mL of final antibody solution19,900
µg/ml0.005
ng/ml5
Final concentration1:250,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:250
µL of 1 µg/mL antibody working stock80
µL of buffer to create 20 mL of final antibody solution19,920
µg/ml0.004
ng/ml4
Final concentration1:500,000
Second dilution factor after preparing 1 µg/mL antibody stock solution1:500
µL of 1 µg/mL antibody working stock40
µL of buffer to create 20 mL of final antibody solution1,960
µg/ml0.002
ng/ml2

Recommended antibody dilutions for Thermo Scientific ECL Substrates

SuperSignal West AttoSuperSignal West DuraSuperSignal West Pico PLUSPierce ECL
Choose when
Detecting very low abundant protein targets, limited antibodies, or limited sample volumesPerforming quantitative western blots or long duration is neededFor routine western blots or new systems not yet optimizedDetecting high abundant protein targets and sample is abundant
Recommended primary antibody dilutions, from a 1mg/mL
1:1,000-1:5,000
(0.2–1 μg/mL)

1:1,000-1:50,000
(0.02–1 μg/mL)

1:1,000- 1:5,000
(0.2–1 μg/mL)

1:100-1:5,000
(0.2–10 μg/mL)

Recommended secondary antibody dilutions, from a 1mg/mL
1:100,000- 1:250,000
(4–10 ng/mL)
1:50,000- 1:250,000
(4–20 ng/mL)
1:20,000-1:100,000
(10–50 ng/mL)
1:1,000-1:15,000
(0.07–1 μg/mL)

Learn why our ECL kits have recommended antibody dilutions

Recommended Alexa Fluor Plus conjugated antibody dilutions for western blotting

Alexa Fluor Plus antibodies
1:5,000- 1:20,000 (0.1 – 0.4 μg/mL)

A note about antibody dilution factors

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.

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