Goat anti-Rabbit IgG (Heavy & Light Chain) Antibody (IRDye680LT)

Details for Product No. ABIN2169666, Supplier: Log in to see
Antigen
Epitope
Heavy & Light Chain
6235
2031
1776
1135
782
648
372
305
34
34
31
15
12
12
12
12
11
7
3
3
1
1
1
1
1
1
1
Reactivity
Rabbit
3081
2914
2380
1662
1509
786
737
489
476
411
401
395
342
298
271
160
159
111
55
34
30
26
16
15
12
10
8
7
5
5
4
4
3
3
3
3
2
2
2
2
2
2
1
1
1
1
1
1
1
Host
Goat
7133
5286
1973
1077
507
362
88
84
51
35
23
15
8
5
3
1
1
Clonality
Polyclonal
Conjugate
IRDye680LT
2227
1823
1751
1266
794
718
430
343
235
147
145
140
140
116
113
112
102
98
94
93
92
91
89
84
81
80
78
75
73
72
68
68
65
61
55
54
54
54
54
53
53
53
53
53
53
52
48
43
41
37
32
31
23
23
22
20
19
18
17
16
15
15
15
14
14
14
12
12
12
12
12
11
11
10
10
10
10
10
9
9
9
9
8
8
8
8
8
8
7
6
6
6
6
6
6
6
5
5
5
5
5
5
5
5
4
4
4
4
3
3
3
3
3
3
3
3
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1
1
1
1
1
1
Application
Immunofluorescence (IF), Immunohistochemistry (IHC), Western Blotting (WB)
Options
Supplier
Log in to see
Supplier Product No.
Log in to see
'Independent Validation' Badge
Lot Number 27397
Method validated Western Blotting
Positive Control MCF-7 cells
Negative Control SK-BR-3 cells
Notes A strong specific band was observed in the positive control at the expected size (~85.5 kDa) that is not observed in the negative control.
Primary Antibody
  • Antigen: Beta Catenin
  • Catalog number: ABIN1043907
  • Supplier: Log in to see
  • Lot number: 27397
  • Dilution: 1:1,000
Secondary Antibody
  • Antibody: IRDye 680LT Goat Anti-Rabbit
  • Catalogue number: Log in to see
  • Supplier: Log in to see
  • Lot number: C30725-01
  • Dilution: 1:10,000
Controls
  • MCF-7 lysates were prepared by ADS Biosystems following standard protocols and quality controlled for protein integrity on a regular basis
  • SK-BR-3 lysates were purchased from Santa Cruz Biotechnology (catalog number sc-2218)
Protocol
  • Lysates were mixed with NuPAGE® LDS Sample Buffer (Life Technologies NP0007) and NuPAGE® Sample Reducing Agent (Life Technologies NP0004) and denatured for 5 minutes at 90ºC.
  • 40 μg of each lysate was electrophoresed on a Bolt 4-12% Bis-Tris Gel (Life Technologies BG04120BOX) and run in Bolt MOPS SDS Running Buffer (Life Technologies B0001) at 160 volts for 1 hour.
  • Odyssey Western Protein Standard (LI-COR #928-40000) was run as a molecular weight standard.
  • PVDF membrane was activated with methanol.
  • Protein samples were transferred to activated PVDF membrane in a wet Bolt Transfer Apparatus (Life Technologies B1000) at room temperature for 1 hour at 20 volts (started at 230mA, ended at 110mA).
  • The membrane was blocked in x LI-COR Odyssey WB block solution for 1 hour at room temperature.
  • The membrane was incubated with the primary antibody diluted 1:1000 in x LI-COR Odyssey WB block solution incubated 2 hours at room temperature.
  • The membrane was washed 4 x 5 minutes in 1 x PBS-T (PBS solution with 0.1% Tween 20).
  • The membrane was incubated with IRDye® 800CW Goat anti-Mouse Secondary Antibody (Red) and IRDye 680LT Goat Anti-Rabbit Secondary Antibody (Green) from LI-COR (#827-11081, Lot #C30725-01), both 1:10,000 dilutions. Incubation was performed at room temperature for 45 minutes.
  • The membrane was washed 4 x 5 minutes in 1 x PBS-T (PBS solution with 0.1% Tween 20).
  • Proteins were detected using Odyssey machine scanning with green channel for loading control and red channel for potential LPL band.
Experimental Notes - No experimental challenges noted.
Validation Images
Western Blotting validation image for anti-Catenin, beta (C-Term) antibody (ABIN1043907) Figure 1: Scanned image of Beta Catenin (Green) and loading control alpha-tubulin (Re...
'Independent Validation' Badge
Lot Number 19538
Method validated Western Blotting
Positive Control A549 cells
Negative Control MCF-7 cells
Notes A strong specific band was observed in the positive control at the expected size (~175 kDa) that is not observed in the negative control.
Primary Antibody
  • Antigen: Epidermal Growth Factor Receptor (EGFR)
  • Catalog number: ABIN98862
  • Supplier: Log in to see
  • Lot number: 19538
  • Dilution: 1:1,000
Secondary Antibody
  • Antibody: IRDye 680LT Goat Anti-Rabbit
  • Catalogue number: Log in to see
  • Supplier: Log in to see
  • Lot number: C30725-01
  • Dilution: 1:10,000
Controls - Lysates were prepared by ADS Biosystems following standard protocols and quality controlled for protein integrity on a regular basis
Protocol
  • Lysates were mixed with NuPAGE® LDS Sample Buffer (Life Technologies NP0007) and NuPAGE® Sample Reducing Agent (Life Technologies NP0004) and denatured for 5 minutes at 90ºC.
  • 40 μg of each lysate was electrophoresed on a Bolt 4-12% Bis-Tris Gel (Life Technologies BG04120BOX) and run in Bolt MOPS SDS Running Buffer (Life Technologies B0001) at 160 volts for 1 hour.
  • Odyssey Western Protein Standard (LI-COR #928-40000) was run as a molecular weight standard.
  • PVDF membrane was activated with methanol.
  • Protein samples were transferred to activated PVDF membrane in a wet Bolt Transfer Apparatus (Life Technologies B1000) at room temperature for 1 hour at 20 volts (started at 230mA, ended at 110mA).
  • The membrane was blocked in x LI-COR Odyssey WB block solution for 1 hour at room temperature.
  • The membrane was incubated with the primary antibody diluted 1:1000 in x LI-COR Odyssey WB block solution incubated 2 hours at room temperature.
  • The membrane was washed 4 x 5 minutes in 1 x PBS-T (PBS solution with 0.1% Tween 20).
  • The membrane was incubated with IRDye® 800CW Goat anti-Mouse Secondary Antibody (Red) and IRDye 680LT Goat Anti-Rabbit Secondary Antibody (Green) from LI-COR (#827-11081, Lot #C30725-01), both 1:10,000 dilutions. Incubation was performed at room temperature for 45 minutes.
  • The membrane was washed 4 x 5 minutes in 1 x PBS-T (PBS solution with 0.1% Tween 20).
  • Proteins were detected using Odyssey machine scanning with green channel for loading control and red channel for potential LPL band.
Experimental Notes - No experimental challenges noted.
Validation Images
Western Blotting validation image for anti-Epidermal Growth Factor Receptor (EGFR) antibody (ABIN98862) Figure 1: Scanned image of EGFR (Green) and loading control alpha-tubulin (Red) Weste...
Immunogen Rabbit IgG
Isotype IgG
Specificity Rabbit IgG
Based on ELISA and flow cytometry, this antibody reacts with the heavy and light chains of rabbit IgG, and with the light chains of rabbit IgM and IgA.
Cross-Reactivity (Details) This antibody was tested by Dot Blot and/or solid-phase adsorbed for minimal cross-reactivity with human, mouse, rat, sheep, and chicken serum proteins, but may cross-react with immunoglobulins from other species.
Purification Immunoaffinity chromatography
Antigen
Application Notes Western blot: 1:20,000 Recommended
Optimum dilutions will vary and should be determined empirically.

NOTE: When using PVDF membranes for Western blotting, SDS (final concentration of 0.1 – 0.2%) and Tween® 20 (final concentration of 0.1 – 0.2%) must be added during the detection incubation step to avoid non-specific background staining.

  • IRDye 680LT secondary antibodies are suitable for immunofluorescent microscopy and other fluorescent imaging applications when using instrumentation with appropriate excitation and detection capabilities.
  • IRDye 680LT is not intended for In-Cell WesternTM assays or in vivo applications.
  • These antibodies are not recommended when getting up and running on system. Once established near-infrared protocols are optimized with IRDye 680RD, IRDye 680LT can be used to optimize signals in the 700 channel. Dilution range 1:20,000 – 1:40,000.
    Note: optimization may be required with IRDye 680LT. Please see pack insert for specific detergents/protocol modifications needed.
  • Comment

    • IRDye 680LT secondary antibodies have been proven to provide the brightest signal for Western blot detection in the 700 nm channel and are comparable to Alexa Fluor® 680 secondary antibodies.
    • Choose IRDye 680LT secondary antibodies to get high signal and for specific uses of detection in the 700nm channel.
    • We do not recommend to start with IRDye 680LT secondaries but rather to start with one of the IRDye 680RD secondaries.

    Restrictions For Research Use only
    Format Lyophilized
    Reconstitution Reconstitute with sterile, distilled water
    Concentration 1.0 mg/mL
    Buffer PBS, pH 7.4
    Preservative Sodium azide
    Precaution of Use This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
    Handling Advice Protect from light.
    Storage 4 °C