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1.
Figure 4

Figure 4. From: Using protein microarray technology to screen anti-ERCC1 monoclonal antibodies for specificity and applications in pathology.

The immunohistochemistry staining on NSCLC tissue sections with rabbit monoclonal anti-PCYT1A antibody or 8F1 mouse monoclonal anti-ERCC1 antibody. Pathologist-validated NSCLC FFPE tissue blocks were cut into thin sections, and then further deparaffinized, re-hydrated before IHC testing. The primary antibodies (Left: rabbit anti-PCYT1A mAb, Right: 8F1 anti-ERCC1 mAb) used for these experiments were diluted at 1:150 dilution with blocking buffer.

Donghui Ma, et al. BMC Biotechnol. 2012;12:88-88.
2.
Figure 7

Figure 7. From: Using protein microarray technology to screen anti-ERCC1 monoclonal antibodies for specificity and applications in pathology.

The immunohistochemistry staining on NSCLC tissue sections with two newly developed mouse monoclonal anti-ERCC1 antibodies. Pathologically-validated NSCLC FFPE tissue sections were used for IHC tests. All the IHC experiments were performed under the same antibody dilution factor (1:150) and incubation condition. The IHC data for two mouse monoclonal antibodies (Left: 4F9; Right: 2E12).

Donghui Ma, et al. BMC Biotechnol. 2012;12:88-88.
3.
Figure 5

Figure 5. From: Using protein microarray technology to screen anti-ERCC1 monoclonal antibodies for specificity and applications in pathology.

qPCR analysis of mRNA expression profile for ERCC1 and PCYT1A genes on 24 lung cancer patient tissues. A. qPCR standard. The experimental data using OriGene’s ERCC1 and PCYT1A qPCR standards. A formula for copy number calculation is shown on the chart. B. qPCR lung cancer tissue expression analysis. qPCRdata on OriGene’s TissueScan lung cancer cDNA array panel (HLRT104). The data were grouped based on cancer patient stages.

Donghui Ma, et al. BMC Biotechnol. 2012;12:88-88.
4.
Figure 3

Figure 3. From: Using protein microarray technology to screen anti-ERCC1 monoclonal antibodies for specificity and applications in pathology.

8F1 immunoblot analyses with purified proteins and antigen absorption test. A. Coomassie staining of purified ERCC1 and PCYT1A proteins. 1ug of affinity purified recombinant ERCC1 and PCYT1A proteins were fractionated on the SDS-PAGE gel and then commassie-stained. B. Immunoblot analysis with 8F1 anti-ERCC1 mAb.0.5ug of purified ERCC1 (Lane 2) and PCYT1A (Lane 4) were loaded on an SDS-PAGE gel and then immunobloted with 8F1 antibody. Empty vector transfected HEK293T cell lysates (Lanes 1 and 3) were used as a negative control. C. Antigen absorption test. Overexpression lysates for PCYT1A (Lane1), ERCC1 (Lane 3), and empty vector transfected control (Lanes 2 and 4) were fractionated on SDS-PAGE, and then immunoblotted with 8F1 (upper panel) or 8F1 pre-depleted with purified PCYT1A protein (lower panels).

Donghui Ma, et al. BMC Biotechnol. 2012;12:88-88.
5.
Figure 1

Figure 1. From: Using protein microarray technology to screen anti-ERCC1 monoclonal antibodies for specificity and applications in pathology.

The production scheme for the protein microarray chip. All the overexpression lysates were produced using OriGene’s TrueORF cDNA clone collection. On a single nitrocellulose slide, over 22,000 protein samples were spotted. These include 10,464 unique gene overexpression lysates printed in duplicate and large selections of positive and negative controls. Since all the expression clones contain a universal Myc and DDK fusion tag, the target gene expression level can be examined by using an Anti-DDK antibody (1:500 dilution of OriGene anti-DDK (TA50011) followed by DyLight 649 conjugated goat anti-mouse IgG secondary antibody for detection.

Donghui Ma, et al. BMC Biotechnol. 2012;12:88-88.
6.
Figure 6

Figure 6. From: Using protein microarray technology to screen anti-ERCC1 monoclonal antibodies for specificity and applications in pathology.

To develop the most highly specific anti-ERCC1 monoclonal antibody with a protein microarray chip. A. Specificity evaluation with a protein microarray chip. The overexpression protein microarray chip was immunostained with the 4F9 clone. This data shows that 4F9 is highly specific to ERCC1 (indicated with red arrows). No cross-reactivity was observed with any other test protein. B. Western Blot confirmation analysis. Seven OriGene VERIFYTM overexpression lysate antigen standards (Lane 1 to 7) were fractionated on SDS-PAGE, and then immunoblotted with 4F9. The recombinant protein expression levels for the each of the different protein overexpression lysates were confirmed with the anti-DDK antibody (Figure 2B). Lanes 1 to 7 are loaded with samples for ERCC1 (NM_202001), ERCC1 (NM_001983), ERCC2 (NM_000400), ERCC3 (NM_000122), ERCC4 (NM_005236), ERCC5 (NM_000123) and PCYT1A (NM_005017).

Donghui Ma, et al. BMC Biotechnol. 2012;12:88-88.
7.
Figure 2

Figure 2. From: Using protein microarray technology to screen anti-ERCC1 monoclonal antibodies for specificity and applications in pathology.

The identification of the 8F1 cross-reactive protein with protein microarray chip and Western blot confirmation. A. Protein microarray hybridization with 8F1. The OriGene overexpression protein microarray chip was immunostained with the most commonly used 8F1 monoclonal anti-ERCC1 antibody. The positive reactive proteins are highlighted with red arrows. These data show that 8F1 recognizes not only its specific target (two ERCC1 transcript variants), but also another unrelated nuclear membrane protein PCYT1A. A number of internal controls were also labeled on the panel. B. Western Blot analysis. Seven OriGene VERIFY™ overexpression lysate antigen standards (Lane 1 to 7) were fractionated on SDS-PAGE, and then immunoblotted with 8F1 (Upper panel). The recombinant protein expression levels within the lysates were analyzed with anti-DDK antibody (Lower panel). Lanes 1 to 7 are loaded with samples for ERCC1 (NM_202001), ERCC1 (NM_001983), ERCC2 (NM_000400), ERCC3 (NM_000122), ERCC4 (NM_005236), ERCC5 (NM_000123) and PCYT1A (NM_005017).

Donghui Ma, et al. BMC Biotechnol. 2012;12:88-88.

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