- PA3-16862 - Invitrogen Antibodies CALR antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of Calreticulin in human kidney lysate using a polyclonal antibody (Product # PA3-16862).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockout validation by Western blot analysis of Calreticulin in lysates of HeLa human cervical epithelial carcinoma parental cell line and Calreticulin knockout (KO) HeLa cell line. Samples were incubated in Calreticulin polyclonal antibody (Product # PA3-16862) using a dilution of 1:1500 followed by a HRP-conjugated Anti-Rabbit IgG secondary antibody. Specific band was detected for Calreticulin at approximately 55 kDa (as indicated) in the parental HeLa cell line, but is not detectable in the knockout HeLa cell line. This experiment was conducted under reducing conditions.

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 # PA3-16862, 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)
Main image
Experimental details: Western blot analysis of Calreticulin in human kidney lysate. Sample was incubated in Calreticulin polyclonal antibody (Product # PA3-16862).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of Calreticulin in 0.5 mg/mL HeLa lysate. Samples were incubated in Calreticulin polyclonal (Product # PA3-16862). This experiment was performed under reducing conditions using the 12-230 kDa separation system..

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 # PA3-16862, 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 # PA3-16862, 1:2000 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 staining of Calreticulin in HCT15 colon cancer cells using Product # PA3-16862. Secondary antibody was an Alexa Fluor 488.

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 # PA3-16862) 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: Immunohistochemical analysis of Calreticulin in formalin-fixed paraffin-embedded tissue section of human thyroid gland. Samples were incubated in Calreticulin polyclonal antibody (Product # PA3-16862) using a dilution of 1:50. The signal was developed using HRP-DAB based detection method which followed counterstaining of the nuclei with hematoxylin. This representative section shows a strong positivity of Calreticulin in the follicular epithelial cells, wherein the signal was found to be very intense in the perinuclear region of the cells which correlates well with Endoplasmic reticulum localization of this protein. The para-follicular cells, endothelial cells of blood vessels (not the RBCs though) and the loose connective tissue in the section showed a weak cytoplasmic staining. Some staining was observed in the follicles/colloids also which is potentially the secreted form of Calreticulin.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemical analysis of Calreticulin in formalin-fixed paraffin-embedded tissue section of human thyroid gland. Samples were incubated in Calreticulin polyclonal antibody (Product # PA3-16862) using a dilution of 1:50. The signal was developed using HRP-DAB based detection method which followed counterstaining of the nuclei with hematoxylin. This representative section shows a strong positivity of Calreticulin in the follicular epithelial cells, wherein the signal was found to be very intense in the perinuclear region of the cells which correlates well with Endoplasmic reticulum localization of this protein. The para-follicular cells, endothelial cells and the loose connective tissue in the section showed a weak cytoplasmic staining. Some staining was observed in the follicles/colloids also which is potentially the secreted form of Calreticulin.

PA3-16862