Summary: Western Blot (WB) run will be optimized to fractionate the purified proteins/cell lysate/ tissue lysate samples on SDS-PAGE followed by transfer of the fractionated sample onto PVDF or nitrocellulose membrane. The membrane is probed with a primary-secondary antibody pair via chemiluminescent or near-infrared (NIR) fluorescence method to detect the fractionated sample of interest. If standard proteins are not transferred, we will repeat the electrophoresis, blotting and probing steps to obtain a satisfactory chemiluminescent or near-infrared (NIR) fluorescence signal. This service often needs to be optimized on a case by case basis to suit the optimal fractionation of protein samples provided by the customer.

Time Line: 5-10 Days.

Number of samples per run: as per request

WB System:

(1) Reducing or Non-Reducing SDS-gel

(2) Linear Gradient (4-20% SDS-PAGE Gel) or Fixed conc. (4, 10, 12% or custom SDS-PAGE Gel)

(3) TRIS-Glycine buffer system or any other system preferred by the customer

(4) Choice of marker/standard proteins

(5) Transfer of proteins on PVDF or nitrocellulose membrane

(6) Probing of the membrane with one primary-secondary antibody pair and visualization by chemiluminescent or near-infrared (NIR) fluorescence method (LI-COR).

Deliverables:

Digital image of the blot.

Requirements:

Customer provides the following

(1) Pure Protein samples in Sample Buffer (>50 µg/protein/run; Conc. = >1mg/ml) or Cell/tissue lysates in Sample Buffer (>500 µg/sample/run; Conc. = >1mg/ml). Alternatively, we may attempt to solubilize the samples in Sample buffer for a nominal fee ($10/pure protein sample).

(2) Primary-secondary antibody pair to detect the protein/fragment of interest.

Available cell lines and tissues:–

HeLa, HEK-293T, H1299, CHO, PC3, SK-N-SH, NIH/3T3

We specialize in all types of ELISAs. We can work with your specific protocol (using your antigens and/or antibodies) or with commercially available kits. We’ve worked with both industry and academic partners.

Capabilities:

• Kit-based (provided by the researcher), custom designed ELISA
• Indirect, Sandwich, competitive
• In-Cell-Western /(ICE)(LI-COR)
• Homogenization/Sonication of cell /tissue lysates
• Transfection/Transduction of mammalian/non-mammalian cells
• Optimization of protocols for commercial kits
• Analyze serum, plasma, cells, tissues
• Analyze fluorescent/chemiluminescent/chromogenic/radioactive substrates

Number of samples processed in a single experiment: 96-well plate format

Typical turn-around time: 7-14 Business days

ELISA with TMB/PNPP substrate or NIR. Primary Antibody, standards or ELISA kit needs to be provided.

Protein expression and purification. Purification of fully functional proteins (with affinity tag) supported by our expertise and instrumentation in assay development.

Expression systems:

• E.coli
• Baculovirus/Insect cell
• mammalian cells
• in vitro expression