Products
Protein Biology Reagents | Consumables
Western Blotting
Blocking Solution
Protein-free Fast Blocking Solution, 500 ml
คุณสมบัติสินค้า:
SKU : G2052-500ML
หมวดหมู่ : Biochemicals , 1. Chemical and Reagents , Buffers , Servicebio ,
แบรนด์ : Servicebio
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Product Information
Product Name | Cat. No. | Spec. |
Protein Free Rapid Blocking Buffer | G2052-500ML | 500 mL |
Product Description/Introduction
This product is a new generation of protein-free fast blocking solution, a mixture of various polymers. which can quickly fill the gaps on the surface of solid support and reduce the interference of non-specific signals; The overall blocking effect is obviously better than the traditional blocking solution based on BSA (bovine serum albumin), skimmed milk powder, casein, etc.; It can be used for blocking and primary antibody dilution in Western blot (WB), ELISA, IHC, IF and other experiments.
Fast and efficient: the blocking time is only 5 minutes, and the signal-to-noise ratio is stronger than that of BSA, skim milk powder, casein and other traditional blocking agents.
Low background: this blocking solution does not contain serum, albumin and other proteins, ensuring a high signal-to-noise ratio.
Good compatibility: Compatible with horseradish peroxidase (HRP), alkaline phosphatase (AP) and biotin labeled secondary antibodies; No preservatives affecting the activity of HRP and AP; does not interfere with biotin-based assays as it does not contain biotin.
Easy to use: This product contains TBST and can be directly used for dilution of primary antibody.
Storage and Shipping Conditions
Ship with wet ice; Store at 2-8; Valid for 12 months; Store at -20 if not used for a long time.
Product Components
Component | G2052-500ML |
Protein Free Rapid Blocking Buffer | 500 mL |
Manuals | One copy |
Assay Protocol/Procedures
1. Membrane blocking in Western blot
a) Wash with TBST for 1-2 minutes after completiing the transfer;
b) According to the size of the membrane, select a suitable container, add a certain volume of protein-free fast blocking solution to ensure cover the membrane completely, and place it on the pendulum or horizontal shaker for 5 minutes (see Question 3 for extended closure if high background occurs);
c) Dilute the primary antibody with protein-free rapid blocking solution, refer to the corresponding primary antibody instructions for dilutions, and make a primary antibody working solution.
d) After the sealing was completed, the protein-free sealing solution was poured out, washed with TBST for 10 seconds, and the primary antibody working solution was added for subsequent incubation.
Note: The primary antibody working solution must completely cover the surface of the PVDF or NC membrane; the main function of the protein-free rapid blocking buffer is to seal the sites on the PVDF or NC membrane that are not bound by proteins, so as to reduce the background, and an increase in the number of bands on the WB exposure may indicate that the antibody recognizes the non-specific bands, and may be replaced by other blocking buffer (milk or BSA);
Western Blot FAQ Reference Table | ||
Problems | Possible causes | Solutions |
1. No signal | 1) Less active HRP on PVDF or NC membranes 2) Mismatch between primary and secondary antibody sources 3) Primary antibody does not recognize the target antigen or has low potency 4) Membrane transfer unsuccessful | 1) Secondary incubation of ECL or extended incubation of ECL with membrane 2) Switch to Ultra Sensitive ECL Exposure 3) Increase sample size 4) Increase the concentration of the primary antibody or switch to a more potent primary antibody, and set up a positive control to ensure that the antibody recognizes the antigen 5) Determine the source of the primary antibody species and select the matching secondary antibody 6) Recommended for electrophoresis and membrane transfer with protein marker to ensure proper electrophoresis and membrane transfer |
2. Rapid signal degradation | 1) ECL deterioration 2) Too much active HRP on PVDF or NC membranes | 1) Replace with a new batch of ECL 2) Reduce sample volume 3) Reduce concentration of primary antibody 4) Reduce secondary antibody concentration |
3. High background | 1) Cross-reactivity of sealers with related reagents 2) incomplete elution 3) Exposure time too long 4) Inadequate blocking | 1) Switch to protein-free rapid blocking buffer 2) Extend the elution time appropriately especially for 0.22 µm PVDF membranes 3) Shorten the exposure time or switch to a less sensitive ECL 4) Extend the blocking time |
4. Brown/yellow bands or bands that are anti-white (ghost bands) | 1) Too much active HRP on PVDF or NC membranes, the oxidatively inactivated portion accumulates in large quantities and becomes visible as a brown/yellow band | 1) Reduce sample volume 2) Reduce concentration of primary antibody 3) Reduce secondary antibody concentration |
5. Non-specific band | 1) Related to antibodies, generally less nonspecific bands for monoclonal antibodies compared to polyclonal antibodies 2) Sample degradation | 1) Choose an antibody with good specificity and high potency 2) Re-take fresh samples |
6. Presence of non-visible white spots within the bands | 1) Bubbles between filter paper and colloid, colloid and membrane, and membrane and filter paper when transferring membrane | 1) When transferring the membrane, be sure to remove the bubbles between the filter paper and the colloid, the colloid and the membrane, and the membrane and the filter paper |
7. Band deformation | 1) Gel inhomogeneity within the gel during gel production 2) Deformation of colloid due to high temperature during electrophoresis 3) Deformation of colloid due to high temperature during transfer 4) Over tightening of clamps during transfer leads to deformation of colloid extrusion | 1) Gel production to ensure gel homogeneity, especially flatness at the interface between the upper and lower gel layers 2) Electrophoresis with lower voltage to reduce heat production or ice bath electrophoresis 3) A full ice bath is required for membrane transfer 4) The clamps should not be too tight or too loose when transferring to ensure that there is a certain amount of pressure can be |
2. Blocking of substrate in ELISA experiment
a) Use antigen or antibody coat ELISA plates
b) Add 300 μL of protein-free rapid blocking solution per well and incubate at 37 for 5 minutes;
c) Continue the following steps such as washing or shaking according to ELISA requirements.
3. blocking in IHC and IF experiments
a) This reagent is used as a solvent to Dissolve 3% (mass to volume ratio) BSA in IHC and IF experiments. It has stable sealing performance and excellent sealing effect in various tissues compared with BSA dissolved in PBS; For antibodies with good specificity, this reagent can be directly used as a blocking agent.
b) Add protein-free rapid blocking agent or BSA dissolved protein-free rapid blocking agent after preparation of the tissue sections,, and leave for 30 minutes at room temperature (with tissue differentiation); Must cover the sample completely to avoid high background from sample drying.
Note
1. No one blocking agent can be applied to all antigen and antibody detection systems. If antibodies unsuitable for this reagent, please replace other blocking agents such as BSA or skim milk powder.
2. Store the remaining solution at 2-8 to avoid pollution after use.
3. This reagent is viscous and should be pipetted slowly to ensure volume accuracy.
4. This product is recommended to be used only once, and repeated use may lead to the decrease of blocking effect; For antibodies with good specificity and high signal-to-noise ratio, the blocking solution can be reused; Do not mix the recovered with the unused blocking solution.
5. For your safety and health, please wear safety glasses, gloves, or protective clothing.
For Research Use Only!
