Esel anti-Ziege IgG (Heavy & Light Chain) Antikörper (IRDye680RD)

Details zu Produkt Nr. ABIN2169629, Anbieter: Anmelden zum Anzeigen
Antigen
Epitop
Heavy & Light Chain
4680
2537
1879
1066
1006
860
298
294
35
34
31
8
7
7
3
3
2
2
1
1
1
1
1
Reaktivität
Ziege
2850
2735
2296
1675
1432
788
613
442
380
379
358
339
291
290
208
138
135
122
41
29
27
25
12
11
9
8
6
5
5
5
4
4
4
4
4
4
4
4
3
3
3
2
2
1
1
1
1
1
Wirt
Esel
6705
4831
1794
891
609
388
91
55
52
21
8
5
5
5
2
1
1
Klonalität
Polyklonal
Konjugat
IRDye680RD
2236
1872
1738
1356
767
682
519
297
264
223
222
200
156
138
128
110
79
75
69
55
51
48
46
43
40
38
34
34
32
29
27
27
27
27
27
27
27
27
22
22
19
19
19
19
15
15
15
15
15
15
14
12
12
10
10
9
9
9
9
9
9
8
8
8
8
8
8
7
6
6
6
6
5
5
5
4
4
4
4
4
4
4
4
4
4
3
3
3
3
3
2
2
2
2
2
2
2
2
2
2
2
2
1
1
1
1
1
1
1
1
Applikation
In-Gel Western blotting (gelWB), Immunohistochemistry (IHC), Western Blotting (WB)
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Hersteller Produkt- Nr.
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'Independent Validation' Siegel
Antigen Goat IgG (Heavy & Light Chain)
Chargennummer C50330-03
Validierte Anwendung Western Blotting
Positivkontrolle Human A375 and SKMEL 28 melanoma cell line lysates; C57BL/6 mouse serum
Primärantikörper ABIN1440014
Sekundärantikörper Donkey anti-Goat IgG (H+L) IRDye680RD (LiCor, antibodies-online product number ABIN2169630 , lot number: C50330-03)
Protokoll
  • 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).
Anmerkungen 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.
Validierungsbilder
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...
Marke In-Cell Western™,CellTag™
Immunogen Goat IgG
Isotyp IgG
Fragment Whole molecule
Spezifität Goat IgG
Based on ELISA, this antibody reacts with the heavy and light chains of goat IgG and with sheep IgG.
Kreuzreaktivität Schaf
Kreuzreaktivität (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.
Produktmerkmale 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.
Reinigung immunoaffinity chromatography
Forschungsgebiet Immunology, Secondary Antibodies
Applikationshinweise Western blot: 1:5,000 - 1:25,000
Beschränkungen Nur für Forschungszwecke einsetzbar
Format Lyophilized
Rekonstitution Reconstitute with sterile, distilled water
Konzentration 1.0 mg/mL
Buffer PBS, pH 7.4
Konservierungsmittel Sodium azide
Vorsichtsmaßnahmen This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
Handhabung Protect from light.
Lagerung 4 °C
Produkt verwendet 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).