HAK21147

IRAK Isoform Antibody Sampler Kit

cat.: HAK21147

Product Type:	Antibody Sampler Kit
Species reactivity:	Human
Applications:	WB

Form:	Liquid
Storage condition:	Store at +4℃ after thawing. Aliquot store at -20℃. Avoid repeated freeze / thaw cycles.
Storage buffer:	1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Concentration:	1ug/ul
Uniprot #:	SwissProt: P51617 Human \| Q62406 Mouse \| O43187 Human \| Q8CFA1 Mouse \| Q4QQS0 Rat \| Q9Y616 Human \| Q9NWZ3 Human \| Q8R4K2 Mouse

Images

Fig1: Western blot analysis of IRAK-1 on different lysates with Rabbit anti-IRAK-1 antibody (HA720079) at 1/2,000 dilution.

Lane 1: A549-si NT cell lysate
Lane 2: A549-si IRAK-1 cell lysate

Lysates/proteins at 10 µg/Lane.

Predicted band size: 77 kDa
Observed band size: 75/77 kDa

Exposure time: 3 minutes; 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 (HA720079) 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.

Fig2: Western blot analysis of IRAK2 on different lysates with Rabbit anti-IRAK2 antibody (ET7108-29) at 1/2,000 dilution.

Lane 1: K-562 cell lysate
Lane 2: PC-12 cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 69 kDa
Observed band size: 69 kDa

Exposure time: 3 minutes; 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 (ET7108-29) 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.

Fig3: Western blot analysis of IRAK4 on different lysates with Rabbit anti-IRAK4 antibody (HA721347) at 1/1,000 dilution.

Lane 1: Jurkat cell lysate
Lane 2: K-562 cell lysate
Lane 3: THP-1 cell lysate

Lysates/proteins at 10 µg/Lane.

Predicted band size: 52 kDa
Observed band size: 52 kDa

Exposure time: 22 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 (HA721347) at 1/1,000 dilution was used in 5% NFDM/TBST 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.

Fig4: IRAK-1 was immunoprecipitated from 0.2 mg HeLa cell lysate with HA720079 at 2 µg/25 µl agarose. Western blot was performed from the immunoprecipitate using HA720079 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: HeLa cell lysate (input)
Lane 2: HA720079 IP in HeLa cell lysate
Lane 3: Rabbit IgG instead of HA720079 in HeLa cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 1 minute; ECL: K1802

	Fig5: Immunohistochemical analysis of paraffin-embedded human colon cancer tissue with Rabbit anti-IRAK-1 antibody (HA720079) at 1/200 dilution. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (HA720079) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
	Fig6: Immunohistochemical analysis of paraffin-embedded human spleen tissue with Rabbit anti-IRAK2 antibody (ET7108-29) at 1/200 dilution. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET7108-29) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
	Fig7: Immunohistochemical analysis of paraffin-embedded human skeletal muscle tissue with Rabbit anti-IRAK4 antibody (HA721347) at 1/1,000 dilution. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (HA721347) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Fig8: Immunocytochemistry analysis of C6 cells labeling IRAK2 with Rabbit anti-IRAK2 antibody (ET7108-29) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 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-IRAK2 antibody (ET7108-29) at 1/100 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 (HA601187, 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.

Fig9: Flow cytometric analysis of RAW264.7 cells labeling IRAK2.

Cells were fixed and permeabilized. Then stained with the primary antibody (ET7108-29, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

Note: All products are “FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE”.