Donkey anti-Goat IgG (Heavy & Light Chain) Antibody (IRDye680RD)

Details for Product No. ABIN2169629, Supplier: Log in to see
Antigen
Epitope
Heavy & Light Chain
6229
2030
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
Goat
3078
2913
2381
1662
1508
785
737
489
476
410
401
395
341
298
271
160
159
111
54
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
Donkey
7130
5285
1970
1077
507
362
89
84
51
35
23
15
8
5
1
1
1
Clonality
Polyclonal
Conjugate
IRDye680RD
2225
1820
1751
1266
793
717
430
343
235
147
145
140
140
116
112
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
In-Gel Western blotting (gelWB), Immunohistochemistry (IHC), Western Blotting (WB)
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'Independent Validation' Badge
Antigen Goat IgG (Heavy & Light Chain)
Lot Number C50330-03
Method validated Western Blotting
Positive Control Human A375 and SKMEL 28 melanoma cell line lysates; C57BL/6 mouse serum
Primary Antibody ABIN1440014
Secondary Antibody Donkey anti-Goat IgG (H+L) IRDye680RD (LiCor, antibodies-online product number ABIN2169630 , lot number: C50330-03)
Protocol
  • WB on HeLa cells and human melanoma cell lines SKMEL28 and A375
    • Mouse serum was collected from C57BL/6 mice and lysed in 6M Urea/20mM Tris.
    • Exosomes were extracted with ExoQuick reagent (SBI, product #EXOQ5A, lot #140624-001) following the manufacturer’s protocol and resuspended in 30µl of modified RIPA buffer.
    • 20µl, 10µl, and 5µl aliquots of 5-fold diluted exosome solution were used.
    • Samples were denatured in Bio-Rad Laemmli sample buffer (product # 1610737, lot #t350001755)containing beta-marcaptoethanol and separated on 4-20% Bio-Rad TGX gel (product #456-1034, lot #t64041496).
    • Transfer onto PVDF membrane (Millipore, product #IPLF00010, lot #R5EA5898E) was done on TransBlot SD apparatus (Bio-Rad) following manufacturer’s protocol.
    • Block membranes with LiCor Blocking Buffer (product 927-40000, lot V1381) for 2h.
    • Incubation with
      • primary CD63 antibody ABIN1440014 at 4°C diluted 1:500 in LiCor Blocking Buffer at 4°C overnight.
      • primary rabbit anti-CD9 antibody (Proteintech, 20597-1-AP, lot 00013334) diluted 1:500 in LiCor Blocking Buffer at 4°C overnight.
    • Wash 4x 15min with PBS-T.
    • Incubation with secondary antibody LiCor Donkey anti-Goat IgG (Heavy & Light Chain) Antibody (IRDye680RD) secondary antibody (antibodies-online product number ABIN2169630, lot C50330-03, 1:15000 dilution) and incubated on a shaker for 1h at RT.
    • Three rinses plus four 15min washes—2 PBS-T, 2 PBS (as required by LiCor).
    • Blot was developed on a LiCor imaging system (Odyssey 9120, scan resolution: 169um, image quality: low).
  • WB on C57BL/6 mouse serum exosomes
    • Mouse serum was collected from C57BL/6 mice and lysed in 6M Urea/20mM Tris.
    • Exosomes were extracted with ExoQuick reagent (SBI, product #EXOQ5A, lot #140624-001) following the manufacturer’s protocol and resuspended in 30µl of modified RIPA buffer.
    • 20µl, 10µl, and 5µl aliquots of 5-fold diluted exosome solution were used.
    • Samples were denatured in Bio-Rad Laemmli sample buffer (product # 1610737, lot #t350001755)containing beta-marcaptoethanol and separated on 4-20% Bio-Rad TGX gel (product #456-1034, lot #t64041496).
    • Transfer onto PVDF membrane (Millipore, product #IPLF00010, lot #R5EA5898E) was done on TransBlot SD apparatus (Bio-Rad) following manufacturer’s protocol.
    • Block membranes with LiCor Blocking Buffer (product 927-40000, lot V1381) for 2h.
    • Incubation with
      • primary CD63 antibody ABIN1440014 at 4°C diluted 1:500 in LiCor Blocking Buffer at 4°C overnight.
      • primary rabbit anti-CD9 antibody (Proteintech, 20597-1-AP, lot 00013334) diluted 1:500 in LiCor Blocking Buffer at 4°C overnight.
    • Wash 4x 15min with PBS-T.
    • Incubation with secondary antibody LiCor Donkey anti-Goat IgG (Heavy & Light Chain) Antibody (IRDye680RD) secondary antibody (antibodies-online, ABIN2169630, lot C50330-03, 1:15000 dilution) and incubated on a shaker for 1h at RT.
    • Three rinses plus four 15min washes—2 PBS-T, 2 PBS (as required by LiCor).
    • Blot was developed on a LiCor imaging system (Odyssey 9120, scan resolution: 169um, image quality: low).
Experimental Notes The donkey anti-Goat IgG (H&L) IRDye680RD-conjugated secondary antibody ABIN2169630 works successfully in Western blot to reveal goat IgG pirmary antibodies used on human cell lysate and prepared mouse serum exosome samples.
Validation Images
Western Blotting validation image for Donkey anti-Goat IgG (Heavy & Light Chain) antibody (IRDye680RD) (ABIN2169630) A. 20µg total protein of cultured HeLa cells and human melanoma cell lines SKMEL28 an...
Brand In-Cell Western™,CellTag™
Immunogen Goat IgG
Isotype IgG
Specificity Goat IgG
Based on ELISA, this antibody reacts with the heavy and light chains of goat IgG and with sheep IgG.
Cross-Reactivity Sheep (Ovine)
Cross-Reactivity (Details) This antibody was tested by Dot Blot and/or solid-phase adsorbed for minimal cross-reactivity with human, mouse, rabbit, rat, chicken, guinea pig, hamster, swine, and horse serum proteins, but may cross-react with immunoglobulins from other species.
Characteristics Blocking buffers and antibody diluents made with bovine serum albumin (BSA) and dry milk may contain IgG that reacts with anti-bovine IgG, anti-goat IgG, anti-horse IgG, and anti-sheep IgG antibodies. This can lead to a significant increase in background and/or reduction of secondary antibody titer for protein detection applications.
Purification immunoaffinity chromatography
Antigen
Application Notes Western blot: 1:5,000 - 1:25,000
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
Product cited in: Alayev, Berger, Kramer, Schwartz, Holz: "The combination of rapamycin and resveratrol blocks autophagy and induces apoptosis in breast cancer cells." in: Journal of cellular biochemistry, Vol. 116, Issue 3, pp. 450-7, 2015 (PubMed).