CCR7 Recombinant Rabbit Monoclonal Antibody [SR36-04] - BSA and Azide free

☑ Knockdown (KD)

All lanes: Western blot analysis of CCR7 with anti-CCR7 antibody (HA750037) at 1:5,000 dilution.
Lane 1/2: Wild-type Hela whole cell lysate (10 µg).
Lane 3/4: CCR7 fragment 1 knockdown Hela whole cell lysate (10 µg).
Lane 5/6: CCR7 fragment 2 knockdown Hela whole cell lysate (10 µg).

HA750037 was shown to specifically react with CCR7 in wild-type Hela cells. Weakened was observed when CCR7 knockdown sample was tested. Wild-type and CCR7 knockdown samples were subjected to SDS-PAGE. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM in TBST for 1 hour at room temperature. The primary antibody (HA750037, 1/500) and Loading control antibody (Rabbit anti-GAPDH, ET1601-4, 1/10,000) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG-HRP Secondary Antibody (HA1001) at 1:200,000 dilution was used for 1 hour at room temperature.

☑ Relative expression (RE)

Western blot analysis of CCR7 on different lysates with Rabbit anti-CCR7 antibody (HA750037) at 1/5,000 dilution.

Lane 1: HDLM-2 cell lysate
Lane 2: Jurkat cell lysate
Lane 3: Daudi cell lysate
Lane 4: MCF7 cell lysate
Lane 5: Mouse spleen tissue lysate
Lane 6: Mouse pancreas tissue lysate (low expression)
Lane 7: Rat spleen tissue lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 43 kDa
Observed band size: 43 kDa

Exposure time: 24 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 (HA750037) at 1/5,000 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 CCR7 on different lysates with Rabbit anti-CCR7 antibody (HA750037) at 1/2,000 dilution.

Lane 1: Raji cell lysate
Lane 2: Daudi cell lysate
Lane 3: EL4 cell lysate
Lane 4: RAW264.7 cell lysate
Lane 5: C6 cell lysate
Lane 6: Rat spleen tissue lysate

Lysates/proteins at 15 µg/Lane.

Predicted band size: 43 kDa
Observed band size: 43 kDa

Exposure time: 10 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 (HA750037) at 1/2,000 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.

Immunocytochemistry analysis of Daudi cells labeling CCR7 with Rabbit anti-CCR7 antibody (HA750037) at 1/250 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-CCR7 antibody (HA750037) at 1/250 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

Immunocytochemistry analysis of RAW264.7 cells labeling CCR7 with Rabbit anti-CCR7 antibody (HA750037) at 1/500 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-CCR7 antibody (HA750037) at 1/500 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

Immunocytochemistry analysis of C6 cells labeling CCR7 with Rabbit anti-CCR7 antibody (HA750037) at 1/200 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-CCR7 antibody (HA750037) at 1/200 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

CCR7 was immunoprecipitated from 0.2 mg Daudi cell lysate with HA750037 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA750037 at 1/2,000 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: Daudi cell lysate (input)
Lane 2: HA750037 IP in Daudi cell lysate
Lane 3: Rabbit IgG instead of HA750037 in Daudi cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 8 seconds; ECL: K1802