AHO1132

antibody from Invitrogen Antibodies

Targeting: HSPB1 CMT2F, Hs.76067, Hsp25, HSP27, HSP28

Antibody data

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Validation data

HSPB1 protein structure - AHO1132 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Extracts prepared from human HeLa cells, mouse 3T3L1 and rat L6 cells were resolved by SDS-PAGE on a 4-20% polyacrylamide gel and transferred to PVDF. The membranes were blocked with a 5% milk-TBST buffer and then incubated with this rabbit polyclonal ant

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: CRISPR-Cas9 mediated genome editing ofHSP27 (as confirmed by next generation sequencing) was achieved by using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR990059_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Fig (a) Western blot analysis of HSP27 was performed by loading 30 µg of HeLa Wild Type (Lane 1), HeLa Cas9 (Lane 2) and HeLa Cas9 cells transduced with HSP27 Lentiviral sgRNA (Lane 3) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-HSP27 Polyclonal Antibody (Product # AHO1132) using 1:1,000 dilution and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036 1:4,000 dilution). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Even though NGS analysis determine the clone as partial KO, there was complete loss of signal in sgRNA transduced cells using the LentiArray™ CRISPR product line confirming that the antibody is specific to HSP27 (Fig (b)).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-HSP27 Polyclonal Antibody(Product # AHO1132) and a 25kDa band corresponding to HSP27 was observed across cell lines tested except HT-1080 which is reported to be negative. Whole cell extracts (30 µg lysate) of MCF7 (Lane 1), MDA-MB-231 (Lane 2), PC-3 (Lane 3), DU 145 (Lane 4), HeLa (Lane 5), LNCaP (Lane 6) and HT-1080 (Lane 7) were electrophoresed using NuPAGE™ 12% Bis-Tris Protein Gel (Product # NP0342BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036,1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).