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

Details zu Produkt Nr. ABIN2169629
Antigen
Epitop
Heavy & Light Chain
1570
134
100
56
34
33
25
6
6
6
6
5
4
4
4
3
2
1
1
1
Reaktivität
Ziege
355
342
337
233
231
122
113
87
80
73
72
65
61
57
54
49
43
31
27
15
9
9
7
5
4
4
4
4
4
3
3
3
3
2
2
1
1
1
1
Wirt
Esel
846
799
335
169
93
84
23
22
13
5
4
4
4
3
1
1
1
1
Klonalität
Polyklonal
Konjugat
IRDye680RD
236
220
209
150
147
130
75
49
48
35
31
26
26
26
26
26
25
23
23
23
22
22
21
21
21
20
20
20
20
20
20
18
16
14
13
10
9
9
9
8
8
8
6
6
6
6
5
5
5
5
5
5
5
5
5
5
4
4
4
4
4
4
4
3
3
3
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1
1
1
1
1
1
1
1
1
1
Applikation
In-Gel Western blotting (gelWB), Immunohistochemistry (IHC), Western Blotting (WB)
Optionen
'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
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
Antigen
Applikations-hinweise 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
Konservierungs-mittel 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).