- PA5-25922 - Invitrogen Antibodies CALR antibody

Submitted references Novel calreticulin-nanoparticle in combination with focused ultrasound induces immunogenic cell death in melanoma to enhance antitumor immunity.

Sethuraman SN, Singh MP, Patil G, Li S, Fiering S, Hoopes PJ, Guha C, Malayer J, Ranjan A
Theranostics 2020;10(8):3397-3412

Low-Intensity Focused Ultrasound Induces Reversal of Tumor-Induced T Cell Tolerance and Prevents Immune Escape.

Bandyopadhyay S, Quinn TJ, Scandiuzzi L, Basu I, Partanen A, Tomé WA, Macian F, Guha C
Journal of immunology (Baltimore, Md. : 1950) 2016 Feb 15;196(4):1964-76

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis in mouse bladder tissue lysates (15 µg per lane) using a CALR polyclonal antibody (Product # PA5-25922).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockout of Calreticulin was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR1007602_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of Calreticulin was performed by loading 30 µg of HeLa wild type (Lane 1), HeLa Cas9 (Lane 2) and HeLa Calreticulin KO (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 Calreticulin Polyclonal Antibody (Product # PA5-25922, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:10,000 dilution) using the iBright™ FL1500 (Product # A44115). Chemiluminescent detection was performed usingSuperSignal™ West Dura Extended Duration Substrate (Product # 34076). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to Calreticulin.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of Calreticulin was achieved by transfecting HeLa cells with Calreticulin specific siRNAs (Silencer® select Product # s114). Western blot analysis (Fig. a) was performed using whole cell extracts from the Calreticulin knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Calreticulin Polyclonal Antibody (Product # PA5-25922, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25µg/ml, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to Calreticulin.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on whole cell extract (30 µg lysate) of RAW 264.7 (Lane 1), SHSY5Y (Lane 2), HeLa (Lane 3), A431 (Lane 4), SKOV3 (Lane 5), IM9 (Lane 6), Jurkat (Lane 7), K562 (Lane 8) and MCF7 (Lane 9). The blot was probed with Anti-Calreticulin Polyclonal Antibody (Product # PA5-25922, 1:1000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/ml, 1:4000 dilution). A 55 kDa band corresponding to Calreticulin was observed in all cell lines tested.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of Calreticulin was performed using 70% confluent log phase U-2 OS cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Calreticulin Polyclonal Antibody (Product # PA5-25922) at 1:200 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing endoplasmic reticulum and mitochondrial localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis in formalin-fixed, paraffin-embedded human colon carcinoma using a CALR polyclonal antibody (Product # PA5-25922), followed by HRP-conjugated secondary antibody and DAB staining.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry analysis of HepG2 cells using a CALR polyclonal antibody (Product # PA5-25922) (bottom) compared to a negative control cell (top) at a dilution of 1:10-50, followed by a FITC-conjugated goat anti-rabbit antibody

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 CRT-NP and FUS local treatment enhanced therapeutic efficacy in vivo and synergized when combined as CFUS. (A) Experimental design to test the efficacy of CFUS against melanoma tumors. (B) Immunofluorescence images of B16F10 melanoma tumor sections showing CRT expression (red) and nuclei (DAPI blue). CFUS significantly enhanced CRT intensity in treated tumors compared to other groups (10X magnification). (C) Growth curves of mice tumors in various experimental groups. CFUS significantly achieved tumor growth delay compared to Control, FUS, and CRT-NPs (n = 4-7). (D) Differences in the survival were determined for each group by the Kaplan-Meier method and the overall P value was calculated by the log-rank test. (E) Representative images of the harvested tumor. (F) Tumor weights at the time of sacrifice showed significant reduction in the overall weight with treatments compared to control. Statistics were determined by ANOVA followed by Fisher's LSD without multiple comparisons correction. * p < 0.05, ** p < 0.01.

PA5-25922

Antibody data