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

Details for Product No. ABIN2169659, Supplier: Log in to see
Antigen
Epitope
Heavy & Light Chain
6012
1904
1869
1159
274
267
266
124
33
30
30
15
12
12
12
12
11
7
3
2
1
1
1
1
1
1
1
Reactivity
Mouse (Murine)
2740
2673
2192
1495
1370
700
664
447
434
362
359
354
275
258
257
147
115
84
43
26
24
21
12
12
8
7
5
5
4
4
4
3
3
3
2
2
2
2
2
2
1
1
1
Host
Goat
6468
4816
1755
887
458
274
87
54
43
35
22
8
6
5
4
1
1
1
Clonality
Polyclonal
Conjugate
IRDye680LT
2064
1741
1639
1144
678
644
422
315
171
122
102
102
94
89
88
88
86
84
79
78
74
73
70
68
68
67
65
65
63
55
55
54
54
54
54
54
53
53
53
53
53
48
47
47
41
40
32
31
30
23
23
22
20
19
18
17
16
15
15
15
14
14
14
12
12
12
12
10
10
10
10
10
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
5
5
4
4
4
4
3
3
3
3
3
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1
1
1
1
1
1
1
Application
Immunofluorescence (IF), Immunohistochemistry (IHC), Western Blotting (WB)
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'Independent Validation' Badge
Antigen Mouse IgG (H&L)
Lot Number C60301-03
Method validated Western Blotting
Positive Control HEK-293 cells transfected with a vector expressing human alpha-sarcoglycan; beta-actin
Notes ABIN2169660 works successfully in Western blot to reveal murine IgG primary antibodies used on HEK-293 cell lysate.
Primary Antibody alpha-sarcoglycan antibody (Leica Biosystems, NCL-L-a-SARC, Lot 6023907) and beta-actin (Sigma-Aldrich, A5441, lot 014M475Y)
Secondary Antibody goat anti-mouse IgG (Heavy & Light Chain) antibody (IRDye680LT)
Protocol
  • Human HEK-293 cells are transfected with a vector expressing human alpha-sarcoglycan and grown in DMEM supplemented with 10% fetal bovine serum at 37°C in a humidified incubator with 5% CO2 to 80% confluency.
  • Lyse cells in lysis buffer (PBS containing 5% sodium deoxycholate and protease inhibitors (Sigma-Aldrich, S8820, lot SLBF3959V).
  • Determine total protein content of the lysates using the bicinchoninic acid assay (Thermo Scientific, 23227, lot RE232692).
  • Denature 5µg, 7.5µg, 10µg, 12.5µg, and 15µg of total protein for 5min at 95°C in 25µl 1x Laemmli buffer and subsequently separate them on a denaturing 10% polyacrilamide gel (Bio-Rad, 161-0183, lot 64063478) for 1h at 120V.
  • Transfer proteins onto nitrocellulose membrane (Bio-Rad, 162-0112, lot A10062318) using a Bio-Rad Trans-Blot Turbo Transfer system for 10min at 1.3A.
  • Cut the membrane into two pieces to detect alpha-sarcoglycan and beta-actin.
  • Block the membrane with TBS containing 5% milk for 1h at RT.
  • Incubate membranes with primary monoclonal alpha-sarcoglycan antibody (Leica Biosystems, NCL-L-a-SARC, Lot 6023907) diluted 1:400 or monoclonal beta-actin (Sigma-Aldrich, A5441, lot 014M475Y) diluted 1:20000 in TBS containing 0.1% tween and 5% milk ON at 4°C.
  • Wash membranes 3x 10min with TBST.
  • Incubate membranes with goat anti-mouse IgG (Heavy & Light Chain) antibody (IRDye680LT) (antibodies-online, ABIN2169660, lot C60301-03) diluted 1:20000 in TBST containing 5% milk for 1h at RT.
  • Wash membranes 3x 10min with TBST.
  • Wash membranes 2x 10min with TBS.
  • Rinse membranes briefly with H2O.
  • Acquire signals with an Odyssey LI-COR CLx imaging system (scan resolution: 169µm, image quality: low) and measure the band intensity using ImageJ.
Experimental Notes The analysis of band intensities show a good linear relationship with the amount of total proteins loaded (R2=0.98). The slope of the lines interpolating the data points of alpha sarcoglycan and beta actin are very similar, allowing a reliable quantification of the expression level of alpha-sarcoglycan after normalizing with beta-actin.
Validation Images
Western Blotting validation image for Goat anti-Mouse (Murine) IgG (Heavy & Light Chain) antibody (IRDye680LT) (ABIN2169660) A. Scanned images of alpha-sarcoglycan and beta-actin Western blot using LI-COR Odyss...
Immunogen Mouse IgG paraproteins
Isotype IgG
Specificity Mouse IgG
Based on ELISA and flow cytometry, this antibody reacts with the heavy and light chains of mouse IgG1, IgG2a, IgG2b, and IgG3, and with the light chains of mouse IgM and IgA.
Cross-Reactivity (Details) This antibody was tested by Dot Blot and/or solid-phase adsorbed for minimal cross-reactivity with human, rabbit, goat, rat, and horse 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
    Supplier Images
    Western Blotting (WB) image for Goat anti-Mouse (Murine) IgG (Heavy & Light Chain) antibody (IRDye680LT) (ABIN2169659) Effect of siRNA and cytokine treatment on phosphorylation of AKT and ERK. The blot wa...
    Product cited in: Mans, Banz, Mueller, Pabst: "The angiogenesis inhibitor vasostatin is regulated by neutrophil elastase-dependent cleavage of calreticulin in AML patients." in: Blood, Vol. 120, Issue 13, pp. 2690-9, 2012 (PubMed).