c-Oncogene Antibody Sampler Kit

Fluorescence multiplex immunohistochemical analysis of mouse hippocampus (Formalin/PFA-fixed paraffin-embedded sections). Panel A: the merged image of anti-GFAP (ET1601-23, Green), anti-NeuN (ET1602-12, Red) and anti-c-Fos (HA722666, White) on hippocampus. HRP Conjugated UltraPolymer Goat Polyclonal Antibody HA1119/HA1120 was used as a secondary antibody. The immunostaining was performed with the Sequential Immuno-staining Kit (IRISKit™MH010101, www.luminiris.cn). The section was incubated in three rounds of staining: in the order of ET1601-23 (1/1,000 dilution), ET1602-12 (1/1,000 dilution) and HA722666 (1/200 dilution) for 20 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 30 mins at 95℃. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Zeiss Observer 7 Inverted Fluorescence Microscope.

Fluorescence multiplex immunohistochemical analysis of mouse brain (Formalin/PFA-fixed paraffin-embedded sections). Panel A: the merged image of anti-GFAP (ET1601-23, Green), anti-NeuN (ET1602-12, Red) and anti-c-Fos (HA722666, White) on brain. HRP Conjugated UltraPolymer Goat Polyclonal Antibody HA1119/HA1120 was used as a secondary antibody. The immunostaining was performed with the Sequential Immuno-staining Kit (IRISKit™MH010101, www.luminiris.cn). The section was incubated in three rounds of staining: in the order of ET1601-23 (1/1,000 dilution), ET1602-12 (1/1,000 dilution) and HA722666 (1/200 dilution) for 20 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 30 mins at 95℃. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Zeiss Observer 7 Inverted Fluorescence Microscope.

Western blot analysis of c-Fos on different lysates with Rabbit anti-c-Fos antibody (HA722666) at 1/1,000 dilution and competitor's antibody at 1/1,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: HeLa serum starved for 40 hours then add 20% FBS for 2 hours cell lysate
Lane 3: RAW264.7 cell lysate
Lane 4: RAW264.7 serum starved for 16 hours then add 200nM PMA for 4 hours cell lysate
Lane 5: C6 cell lysate
Lane 6: C6 serum starved for 16 hours then add 10% FBS for 30 minutes cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 41 kDa
Observed band size: 41-55 kDa

Exposure time: Lane 1-6 (left): 3 minutes; ECL: K1801;
Exposure time: Lane 1-6 (right): 1 minute 2 seconds; ECL: K1802;
4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA722666) at 1/1,000 dilution and competitor's antibody at 1/1,000 dilution were used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells with c-Myc (HA721182) or Normal Rabbit IgG according to the ChIP protocol. The enriched DNA was quantified by real-time PCR using indicated primers. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.

Western blot analysis of Ras on different lysates with Rabbit anti-Ras antibody (HA721883) at 1/1,000 dilution.

Lane 1: HEK-293 cell lysate (20 µg/Lane)
Lane 2: Jurkat cell lysate (20 µg/Lane)
Lane 3: SH-SY5Y cell lysate (20 µg/Lane)
Lane 4: MCF7 cell lysate (20 µg/Lane)
Lane 5: A431 cell lysate (20 µg/Lane)
Lane 6: A375 cell lysate (20 µg/Lane)
Lane 7: C6 cell lysate (20 µg/Lane)
Lane 8: NIH/3T3 cell lysate (20 µg/Lane)
Lane 9: Rat spleen tissue lysate (40 µg/Lane)
Lane 10: Mouse spleen tissue lysate (40 µg/Lane)

Predicted band size: 21 kDa
Observed band size: 21 kDa

Exposure time: 25 seconds;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA721883) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Western blot analysis of Src on different lysates with Rabbit anti-Src antibody (ET1609-65) at 1/1,000 dilution.

Lane 1: A549-WT cell lysate
Lane 2: A549-KD Src cell lysate

Lysates/proteins at 10 µg/Lane.

Predicted band size: 56 kDa
Observed band size: 54 kDa

Exposure time: 20 seconds; ECL: K1801;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1609-65) at 1/1,000 dilution was used in 5% BSA at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

c-Myc was immunoprecipitated from 0.2 mg HeLa cell lysate with HA721182 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA721182 at 1/1,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: HeLa cell lysate (input)
Lane 2: HA721182 IP in HeLa cell lysate
Lane 3: Rabbit IgG instead of HA721182 in HeLa cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 30 seconds; ECL: K1801