Western Blot (WB) is considered to be the most sensitive, simple, and unequivocal immunodetective application available. This is due to the high amount of information obtained upon performance, such as target molecular weight and quantitative, as well as qualitative, information of the protein of interest.
The Human Protein Atlas (HPA) project 1,2 has been set up to allow for a systematic exploration of the human proteome using Antibody-Based Proteomics. This is accomplished by combining high-throughput generation of Prestige Antibodies® with protein profiling in a multitude of human tissues and cells. The standard evaluation of the Prestige Antibodies® is immunochemical staining (IHC, ICC), but all antibodies are also tested in WB and Immunofluorescence (IF). The resulting IHC, WB, ICC, and IF images are publicly available on the Human Protein Atlas web portal.3 Each year, protein expression and localization data of approximately 2,500 new proteins are added to the portal. By 2015, a first draft of the localization of the full human proteome is expected to be available.
The Prestige Antibodies® are tested in WB (Figure 1) in a standardized sample set-up using total protein lysates. Testing is done in a single attempt and the resulting blot images are shown on the HPA portal. For the majority of the Prestige Antibodies®, the protein sample set-up in HPA is lysate of liver tissue, tonsil tissue, urinary bladder transitional cell carcinoma cell line RT-4, glioblastoma cell line U-251MG, and IgG/HSA-depleted human plasma. The entire blot with all five sample lanes is presented, regardless of whether the lanes show single bands, no bands or multiple bands.
Figure 1.Schematic figure of the WB procedure used within the Human Protein Atlas project. By electrophoresis, proteins are separated according to molecular weight. After electro-transfer and immobilization on membrane, Prestige Antibodies® are used as primary antibodies for detection of target proteins. Antibody binding is visualized by chemiluminescence detection in a CCD-camera system using a horse radish peroxidase (HRP) labelled secondary antibody.
Failure in detection, i.e. no or only background detection, may not necessarily be related to a technical problem, but rather to biological features of the analyzed protein. For example, the target protein may be solely expressed during short processes or stages in the cell or only present in specific cells and tissues. The protein may also be present in amounts too low to detect.
The migration of a protein through a gel is affected by other factors than the size of the protein. Such factors include:
The performance of the Prestige Antibodies® in WB is validated in HPA by a score system, resulting in supportive, uncertain, and not supportive assignments. Supportive results correspond to achieved bands of predictive size with, or without, additional bands present (Figure 2 and 3). Uncertain results correspond to empty blots (no bands) or to single bands differing more than +/- 20% from predicted sizes in kDa. Finally, not supportive results refer to blots with bands not corresponding to predicted sizes or to weak bands of predicted sizes, but with additional bands of higher intensity also present.
On the HPA, WB images scored as supportive and uncertain are shown, providing free assessment of the data. WB images scored not supportive are not presented.
Figure 2.Examples of supportive WB images showing single bands corresponding to predicted sizes in kDa (+/-20%).
A) HPA000898, Anti-HSPA9, target weight: 74 kDa. Lane 1: Marker [kDa]: 206,113,82,49,32,26,17.8; Lane 2: RT-4; Lane 3: U-251MG sp; Lane 4: A-431; Lane 5: Liver; Lane 6: Tonsil.
B) HPA007292, Anti-SLC27A5, target weight: 75, 67 kDa. Lane 1: Marker [kDa]: 230,130,95,72,56,36,28,17,11; Lane 2: RT-4; Lane 3: U-251MG sp; Lane 4: Human Plasma; Lane 5: Liver; Lane 6: Tonsil.
Figure 3.Examples of supportive WB images showing bands of predicted sizes in kDa (+/-20%) with additional bands present.
A) HPA002877, Anti-PGRMC1, target weight: 22 kDa. Lane 1: Marker [kDa]: 206,113,82,49,32,26,17.8; Lane 2: RT-4; Lane 3: U-251MG sp; Lane 4: A-431; Lane 5: Liver; Lane 6: Tonsil.
B) HPA004125, Anti-MARS, target weight: 101, 49 kDa. Lane 1: Marker [kDa]: 230,110,82,49.3,32.2,25.5,17.6; Lane 2: RT-4; Lane 3: U-251MG sp; Lane 4: Human Plasma; Lane 5: Liver; Lane 6: Tonsil.
Ongoing testing in over-expressed lysates (Origene Technologies) for antibodies previously scored as uncertain or not supportive using total protein lysates, results in a supportive score for 90% of the analyses.
Protein samples (selected tissue lysates, cell lysates or over-expression lysates) are mixed with Laemmli buffer (to a final loading concentration of 2% SDS, 10% glycerol, 0.002% bromophenol blue, 0.0625 M TrisHCl), supplemented with DTT to a final concentration of 50 mM, and incubated in 95 °C for 5 minutes.
All incubation and wash steps are performed at room temperature and with agitation.
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