VERIFY Tagged Antigen. Validation Data

Transcription

1 VERIFY Tagged Antigen Validation Data

2 Antibody Validation Figure 1. Over-expression cell lysate for human STAT3 (NM_139276) was used to test 3 commercial antibodies. Antibody A shows strong antigen binding. Antibody C shows weak binding, while Antibody B did not react with STAT3 at all A B C Figure 2. Over-expression cell lysate for human LRRK2 (NM_198578) was used to validate two anti-lrrk2 antibodies in two rounds of Western blot experiments. The left panel shows positive reaction with a commercial monoclonal antibody. The right panel shows a negative reaction with a polyclonal antibody generated against a protein peptide TFX1 TFX2 vec Lysate Lysate vec TFX1 TFX2 vec Lysate Lysate vec

3 Antibody Validation P53 Ab kda 188 Vendor A ***** Vendor B *** Vendor C * Vendor D **** Vendor E Figure 3. Five commercial antibodies against human P53 were evaluated in Western blot experiments with P53 over-expression cell lysate. P53 protein level in cell lysate was pre-determined using a purified GST-Myc-DDK standard. Lysate was serially diluted before SDS-PAGE and immunoblotting. Antibodies A-C are rabbit antibodies. D and E are mouse antibodies. Antibody quality and star rating is based on P53 protein detection level.

4 Determining Consistency kda Antibody Lot A Antibody Lot B Antibody Lot C Figure 4. Three different lots of a commercial polyclonal antibody against human P53 were evaluated in Western blot experiments with P53 over-expression cell lysate. One can see certain lot to lot variation in amount of antigen detection, especially between lot A and C.

5 Endogenous Protein Expression Level P53-myc-DDK 10ug/lane ng 10ng 5ng 1ng 0.5ng A431 HT Anti-p Figure 5. Estimate the endogenous p53 level in different carcinoma cell line by VERIFY antigen standard. 10 ug whole cell lysates from 3 different cancer cell lines were tested against titrated p53 over-expression lysate. Using overexpression lysate as standard, it is determined endogenous p53 expression level in these 3 cell lines were 3.8 ng (A431), 0.4 ng (HT-29), and 1.3 ng (HEKT293) respectively.

6 Over-expression Cell Lysate QC Figure 6-8 show typical Western blot images when over-expression cell lysates were tested with anti-tag antibodies. Figure 4 shows a comparison using either anti-myc antibody (TA100010) or anti-ddk antibody (TA100011). Figures 5 and 6 were done with anti-myc antibody. Most proteins are detected as a single band. Some proteins are detected as two or multiple bands due to various reasons, such as post-translation modification, protein degradation, etc. Figure 6

7 Over-expression Cell Lysate QC Figure 7 Figure 8

8 Lysate Stability Test Figure 9. Real time stability study was run on two different cell lysates, one transmembrane protein (SIGLEC9), the other cytosolic protein. When in SDS buffer, both lysates exhibited protein integrity at room temperature for up to when tested in Western blots with anti-myc antibody (TA10010). Also, up to 5 freeze-thaw cycles does not show significant protein degradation. All over-expression cell lysates are shipped as lyophilized powder. Upon receiving, we recommend storage at -20 C, either as lyophilized or after resuspension in SDS buffer. SIGLEC9 PIP5KIC KDa cycles F/T R/T 4C -20C 5 cycles F/T R/T 4C -20C

9 Lysate Stability Test Figure 10. Stability of lyophilized over-expression cell lysates. Two over-expression cell lysate samples were lyophilized in RIPA buffer overnight, and then reconstituted in SS buffer and tested in Western blot with anti-myc antibody (TA100010). No significant difference was detected among all tested samples. : original lysate before lyophilization Lane 1: lyophilized and then reconstituted in SDS sample buffer immediately Lane 2: lyophilized, and then stored at 37 C for one day before reconstitution in SDS sample buffer. PIP5KIC SIGLEC9 PIP5KIC SIGLEC9 KDa Ponceaus staining after transfer Anti-myc IB

10 Other Applications Tagged Over-expression Cell Lysates can potentially be used in many other applications, such as antibody production, immunoprecipitation, proteinprotein interaction, etc.. Figure 9 shows a preliminary protein array results when spotted lysate arrays are detected using antimyc and anti-ddk antibodies each labeled with different fluorescent dyes (Alexa-555 and Alexa-647). Top two panels are individual antibody staining, while bottom panel shows combined image to illustrate differential binding. Protein arrays can be used in detection of autoimmune antibodies, and antibody decoding.