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Blocking Buffer for Fluorescent Western Blotting

Product Sizes
125 ml
MB-070S-125ML
About this Product
SKU:
MB-070S
Additional Names:
Multiplex Blocking Buffer, Fluorescent Blocking Buffer, Blocking Solution, Blocking Buffer Western Blot, IRDye Western Blot Blocking Buffer, Alexa Dye Blocking Buffer, DyLight Blocking Buffer
Application:
Cell-based/Functional Assay, ELISA, Immunofluorescence, Immunohistochemistry, Immunoprecipitation, Microarray, Western Blot
Buffer:
See application note.
CE/IVD:
RUO
Concentration:
1X
Extra Details:
This blocking buffer is specifically designed for Western blotting using fluorochrome conjugated antibodies. Pure nitrocellulose membrane is recommended for maximum performance. Other membranes, such as PVDF or nitrocellulose embedded in a support can be used, but may generate elevated backgrounds. Protein should be transferred from gel to membrane using standard protocols. Blocking buffer can be used for membrane blocking and to dilute both primary and secondary antibodies. Blocking Buffer for Fluorescent Western Blotting is suitable for use with imaging systems produced by Bio-Rad Laboratories, GE Healthcare, Alpha Innotech, FujiFilm Life Science, Licor Biosciences, UVP and Syngene.
Physical State:
Liquid
Shipping Conditions:
Blue Ice
Specificity:
Blocking buffer was prepared using ultra pure reagents dissolved in pharmaceutical grade water (WFI) and consists of a proprietary protein formulation in TRIS buffered saline at pH 7.6 with thimerosal added as an antimicrobial agent.
Storage Conditions:
2-8[o]C unopened. Do not freeze.
Supplier:
Rockland Inc
Type:
Buffers & General Consumables: Others
Comparison of the performance of different blocking reagents in epitope mappings with PEPperCHIP® Peptide Microarrays. The PEPperCHIP® Peptide Microarrays were blocked for 30 minutes with either 2% skim milk powder (A), 1% HSA (B), 1% BSA (C) or 100% Rockland Blocking Buffer [p/n MB-070] (D). A human serum sample was assayed at dilution 1:200, followed by detection with secondary goat anti-Human IgG (H+L) DyLight™ 680 Antibody [p/n 609-144-123] and a control anti-HA (12CA5)-DyLight™ 800 Antibody. Red spots = sample IgG response and frame of polio control peptides, green spots = frame of HA control peptides.
Selected sections of the PEPperCHIP® Peptide Microarrays after assay with different blocking reagents. The microarrays were blocked for 30 minutes with either 2% skim milk powder (A), 1% HSA (B), 1% BSA (C) or 100% Rockland Blocking Buffer [p/n MB-070] (D), respectively. A human serum sample was assayed at dilution 1:200, followed by detection with secondary goat Anti-Human IgG (H+L) DyLight™ 680 Antibody [p/n 609-144-123]. Red spots = sample responses and polio control peptides, green spots = HA control peptides. The underlying binding motifs of the respective sections are indicated on the left.
Dot Blot of Human IgA Fluorescein using MB-070. Antigen: Human IgA Fluorescein. Load: 3-fold serial dilution starting at 200 ng. Block: MB-070 for 30 min at RT.
Western Blot of Fluorescent TrueBlot®: Anti-Rabbit IgG DyLight 680 Conjugated using MB-070. Lane 1: Rabbit IgG, Non-denatured. Lane 2: Rabbit IgG, Denatured. Load: 50 ng per lane. Primary antibody: none. Secondary antibody: Fluorescent TrueBlot®: Anti-Rabbit IgG DyLight 680 Conjugated antibody at 1:1,000 for 60 min at RT. Block: MB-070 for 30 min at RT. Predicted: 160 kDa for non-denatured; observed: 170-180 kDa for non-denatured. Band migrates at slightly higher molecular weight.
Multiplex western blot results using MB-070. Rockland Mouse-a-GST (200-301-200 lot 24882, blue), Rabbit anti-Transferrin (109-4134 lot 3033), and Goat-anti-Alpha-1-Anti-Trypsin (100-101-147 lot 5842) were used in a multiplex system to detect target proteins under reducing conditions in albumin depleted human serum with 320 ng of added GST. Sample was run by SDS-PAGE, transferred to 0.2 um PVDF using the BioRad Trans-Blot Turbo and blocked in 2.5% Blotto, 2.5% BSA, 0.02% Tween over night at 4°C. Membrane was probed with three primary antibodies at 1:1000 dilution (in MB-070 over night at 4°C). Detection shown was using DyLight™549 Donkey anti-Rabbit IgG (611-742-127 lot 21100, shown as green) DyLight™488 Donkey anti-Mouse IgG (610-741-124 lot 21095, shown as blue), and DyLight™649 Donkey anti-Goat IgG (605-743-125 lot 20834, shown as red) at 1:10,000 (in MB-070 at 30 min RT). Blots were washed, rinsed in methanol, dried and Images were collected using the BioRad VersaDoc System.