Choosing the right reagents and accessories for Western blotting is crucial for achieving clear, reliable results. The quality and compatibility of these components directly impact the specificity and sensitivity of your assays, making it essential to select them wisely to ensure successful experiments.
Western blotting, a cornerstone technique in molecular biology and biochemistry, hinges on the precise detection of proteins. This method's success relies heavily on the seamless interaction between various reagents, including protein markers, blocking buffers, antibodies, transfer buffers, and detection reagents.
Western Blot Essentials
Sample Buffers and Controls
Sample buffers are essential for preparing protein samples for electrophoresis, ensuring they are in an optimal state for separation. They play a critical role in denaturing proteins, reducing disulfide bonds, and loading samples uniformly. Control lysates, on the other hand, are crucial for verifying the effectiveness of immunodetection and transfer processes.
| Product | Application Note |
| 2X SDS-PAGE Sample Buffer | Sample buffers contain SDS to denature proteins and impart a uniform negative charge, enabling size-based separation. |
| 2X SDS-PAGE Sample Buffer without DTT or b-ME | Denaturing buffer without reducing agents (DTT or b-ME). Sample buffers contain SDS to denature proteins and impart a uniform negative charge, enabling size-based separation. |
| 12 Epitope Tag Control Lysate (GST) | Contains GST--T7--HSV--cMyc--VSV--Glu-Glu--V5--E-tag--Flag--S tag--HA--6XHis. Proteins tagged with multiple epitope tags offer a reliable benchmark for evaluating protein transfer and facilitating accurate immunodetection. |
| 12 Epitope Tag Control Lysate (MBP) | Contains MBP--T7--HSV--cMyc--VSV--Glu-Glu--V5--E-tag--Flag--S tag--HA--6XHis. Proteins tagged with multiple epitope tags offer a reliable benchmark for evaluating protein transfer and facilitating accurate immunodetection. |
| DYKDDDDK (FLAG®) Positive Control Lysate | Ready-to-use. Supplied in denaturing buffer without reducing agents (DTT or b-ME). FLAG®-Tag control lysate can be used to evaluate protein transfer and accurate immunodetection using anti-flag tag antibodies. |
| DYKDDDDK (FLAG®) Positive Control Lysate | Ready-to-use FLAG®-Tag control lysate can be used to evaluate protein transfer and accurate immunodetection using anti-flag tag antibodies. |
| Ponceau S Solution | Ponceau S staining can be employed post-transfer to provide a rapid and reversible method for assessing overall protein transfer efficiency. |
Protein Markers
Protein markers are essential for estimating protein sizes in SDS-PAGE and Western blotting, acting as references to compare against unknown proteins. Prestained markers allow real-time tracking and immediate verification of protein transfer, eliminating the need for additional staining. Unstained markers, requiring staining post-electrophoresis, provide precise molecular weight estimates and are versatile for different staining methods. Choose based on your experiment's need for tracking versus accuracy.
| Product | Molecular Weight Range | Number of Bands | Stain |
| Protein Molecular Weight Marker | 14-120 kDa | 7 | Unstained |
| Sapphire Prestained Protein Standard 10-180kDa | 10-180 kDa | 11 | Prestained, blue |
| Opal Prestained Protein Standard 10-180kDa | 10-180 kDa | 10 | Prestained, three colors |
| Opal Prestained Protein Standard 10-245kDa | 10-245 kDa | 12 | Prestained, three colors |
| Ruby Prestained Protein Standard 10-175kDa | 10-175 kDa | 11 | Prestained, two colors |
| Blue MW Ladder Antibody Peroxidase Conjugated | - | - | Useful for HRP-based luminescent detection. Compatible with Sapphire Prestained Protein Standard 10-180kDa |
Assay Buffers
Assay buffers are essential for efficient SDS-PAGE, ensuring optimal protein transfer in Western blotting, and facilitating membrane reprobing during immunodetection.
| Product | Application Note |
| 10X SDS-PAGE Running Gel Buffer | 10X concentrated stock solution. |
| 10% Sodium Dodecyl Sulfate (SDS) | Concentrated stock solution. Should be diluted appropriately with distilled, deionized water or equivalent to its final working concentration. |
| 20% Sodium Dodecyl Sulfate (SDS) | Concentrated stock solution. Should be diluted appropriately with distilled, deionized water or equivalent to its final working concentration. |
| 1X SDS-PAGE Destain Solution | Uniquely formulated, ready-to-use reagent specifically designed for destaining Coomassie-stained polyacrylamide gels. |
| Express Transfer Buffer | Alternative to tris-glycine as a transfer buffer in Western blot. It may run at an elevated temperature but gives noticeably lower background. |
| Revitablot™ Western Blot Stripping Buffer | Useful for repeated stripping of antibodies from membranes for reprobing purposes. |
Western Blot Blocking Buffers
Blocking buffers play a critical role in Western blotting by coating the membrane to prevent non-specific binding of detection antibodies, thereby enhancing the specificity and clarity of the signal.
| Product | Application Note | Precautions |
| Blocking Buffer for Fluorescent Western Blotting | Ready-to-use solution. Designed for use in fluorescent Western blotting. | Contains thimerosal, for a thimerosal-free option please see Blocking Buffer (10X) for Fluorescent Western Blotting (Thimerosal Free). |
| Blocking Buffer (2X) for Fluorescent Western Blotting | 2X concentrated solution. Designed for use in fluorescent Western blotting. | Contains thimerosal, for a thimerosal-free option please see Blocking Buffer (10X) for Fluorescent Western Blotting (Thimerosal Free). |
| Blocking Buffer for Fluorescent Western Blotting (Thimerosal Free) 10-Pack | 10-pack of ready-to-use solution. Thimerosal-free blocking buffers are often preferred in facilities where safety and environmental impact are significant considerations. | |
| Blocking Buffer (10X) for Fluorescent Western Blotting (Thimerosal Free) | 10X concentrated solution. Thimerosal-free blocking buffers are often preferred in facilities where safety and environmental impact are significant considerations. | |
| BlockOut® Universal Blocking Buffer for Western Blotting | Universal blocking buffer for Western blotting. Recommended when phospho-specific antibodies are used. | |
| BlockOut® (2X) Universal Blocking Buffer for Western Blotting | 2X concentrated solution. Universal blocking buffer for Western blotting. Recommended when phospho-specific antibodies are used. | |
| Normal Goat Serum (NGS) | For use in immunoassays where the primary antibody was produced in goat. | |
| Normal Horse Serum (NHS) | For use in immunoassays where the primary antibody was produced in horse. | |
| Normal Mouse Serum (NMS) | For use in immunoassays where the primary antibody was produced in mouse. | |
| Normal Rabbit Serum (NRS) | For use in immunoassays where the primary antibody was produced in rabbit. | |
| Normal Rat Serum (NRS) | For use in immunoassays where the primary antibody was produced in rat. | |
| Normal Sheep Serum (NSS) | For use in immunoassays where the primary antibody was produced in sheep. | |
| Bovine Serum Albumin 30% Solution | BSA blocking solutions are preferred with biotin and AP antibody labels. | |
| Bovine Serum Albumin - Fraction V | BSA blocking solutions are preferred with biotin and AP antibody labels. | Since BSA is notoriously difficult to dissolve, you can save time using our Bovine Serum Albumin 30% Solution and dilute further. |
| Blotto Immunoanalytical Grade (Non-Fat Dry Milk) | Immunoanalytical grade non-fat dry milk. | Not suitable for use with biotin-avidin detection due to inherent biotin content. |
| Blotto A Pre-Mixed | General purpose blocking agent. | |
| Blotto B Pre-Mixed | General purpose blocking agent when phospho-specific antibodies are used. | |
| 10X BBS Fish Gel Concentrate | Fish gel is less likely to cross-react with antibodies of mammalian origin than conventional blocking reagents such as non-fat dry milk and BSA. | |
| 10X PBS Fish Gel Concentrate | Fish gel is less likely to cross-react with antibodies of mammalian origin than conventional blocking reagents such as non-fat dry milk and BSA. | For AP antibody labels use 10X TBS Fish Gel Concentrate or 10X BBS Fish Gel Concentrate buffers instead. |
| 10X TBS Fish Gel Concentrate | Fish gel is less likely to cross-react with antibodies of mammalian origin than conventional blocking reagents such as non-fat dry milk and BSA. | |
| 1X PBS, pH 7.2 Buffer with 1% Casein | Blocking buffers containing casein may provide lower backgrounds than buffers containing non-fat milk or BSA. Casein is recommended for applications using biotin-avidin complexes. | For AP antibody labels use 1X TBS, pH 7.8 Buffer with 1% Casein instead. |
| 1X TBS, pH 7.8 Buffer with 1% Casein | Blocking buffers containing casein may provide lower backgrounds than buffers containing non-fat milk or BSA. Casein is recommended for applications using biotin-avidin complexes. | |
| Blocking Buffer Sampler Kit | Set of different blocking buffers for all sorts of immunoassays. |
Western Blot Substrates
In Western blotting, substrate selection is guided by the enzyme label. Luminescent substrates, compatible with horseradish peroxidase (HRP), offer unparalleled sensitivity for low-abundance proteins, necessitating specialized imaging tools. Conversely, chromogenic substrates work well with alkaline phosphatase (AP) and HRP labels alike, providing a user-friendly, equipment-free visualization of results.
| Product | Detection Type | Compatible Label |
| BCIP/NBT Membrane Alkaline Phosphatase Substrate | Chromogenic | Alkaline phosphatase (AP) |
| BCIP/TNBT Membrane Alkaline Phosphatase Substrate | Chromogenic | Alkaline phosphatase (AP) |
| Chemiluminescent FemtoMax™ Super Sensitive HRP Substrate | Luminescent | Peroxidase (HRP) |
| DAB Membrane Peroxidase Substrate (50X) | Chromogenic | Peroxidase (HRP) |
| TMB Membrane Peroxidase Substrate - Ready-To-Use | Chromogenic | Peroxidase (HRP) |
| UniGlow™ - One Component Chemiluminescent Substrate | Luminescent | Peroxidase (HRP) |
