Little knowledge of blocking solution in ELISA experiment
The description of the ELISA kit says that the blocking solution is sucrose solution. Generally, BSA or casein protein molecules are used to block blank sites. The principle of sucrose blocking is:
1. In the ELISA experiment, sucrose itself has no blocking effect;
2. The blocking solution is generally BSA, casein etc. or Tween etc.
3. The main purpose of adding sucrose is to protect the protein adsorbed (coated) by the ELISA board and play the role of "protective agent".
4. The sucrose solution is generally added after the ELISA plate is blocked by BSA. Of course, sucrose is also added to the blocking solution for processing at the same time; which is better requires your experiment to verify later. But most choose the former.
The blocking agent can also use 5% skimmed milk (commercially available), 0.4% gelatin, but experiments show that the former is better, the latter is not easy to clean the unbound gelatin.
The principle of the blocking agent is to block the microwells of the enzyme-linked plate without binding antigen or antibody with blocking agent. When binding the antibody or antigen, no non-specific binding will occur, and the accuracy of the experiment will not be affected.
The role of tween: Although it cannot be blocked separately, it will play the role of washing non-specifically bound or weakly bound proteins during blocking, if the antibody is coated on the plate, YY-
Attachment: Configuration of reagents such as buffer solution in ELISA experiment
The ELISA experiment is based on the immune reaction of antigens and antibodies. How to configure the ELISA reagents and various solutions? Recently, the ELISA experiment has just been done. The various ELISA configuration methods are summarized as follows: 2) ELISA experiment washing buffer (2) ELISA experiment washing buffer (3) ELISA experiment diluent; (4) ELISA experiment termination solution (5) ELISA substrate buffer (6) ELISA experiment TMB (tetramethylbenzidine ) Use solution (7) ELISA experiment ABTS use solution (8) ELISC.
Reagent
(1) ELISA test coating buffer (PH9.6 0.05M carbonate buffer):
NaHCO3 1.59g
NaHCO3 2.93g
Add distilled water to 1000ml
(2) ELISA experiment washing buffer (PH7.4 PBS): 0.15M
KH2PO4 0.2g
Na2HPO4 · 12H2O 2.9g
NaCl 8.0g
KCl 0.2g
Tween-20 0.05% 0.5ml
Add distilled water to 1000ml
(3) ELISA experiment diluent:
Bovine serum albumin (BSA) 0.1 g
Add wash buffer to 100ml
Or use sheep serum, rabbit serum and other serums to mix with 5-10% of the washing solution.
(4) ELISA experiment termination solution (2M H2SO4):
Distilled water 178.3ml, 21.7ml of concentrated sulfuric acid (98%) was added dropwise.
(5) ELISA substrate buffer (PH5.0 phosphate date citric acid):
0.2M Na2HPO4 (28.4g / L) 25.7ml
0.1M citric acid (19.2g / L) 24.3ml
Add 50ml of distilled water.
(6) ELISA experiment TMB (tetramethylbenzidine) using solution:
TMB (10mg / 5ml absolute ethanol) 0.5ml
Substrate buffer (PH5.5) 10ml
0.75% H2O2 32μl
(7) ELISA experiment ABTS solution:
ABTS 0.5mg
Substrate buffer (PH5.5) 1ml
3% H2O2 2μl
(8) ELISA experiment blocking solution:
Bovine Serum Albumin (BSA) 5g
Add wash buffer to 100ml.
It is worth mentioning that some blocking fluids contain skimmed milk powder, which is not suitable for long-term storage.
The biological gang recommends ELISA-related products. For details, see: blocking reagent, termination solution, blocking peptide, ELISA diluent, ELISA kit, ELISA buffer, ELISA elution buffer
In order to ensure the accuracy of the ELISA experiment, the buffer and each component added in the experimental operation must be accurate. When the washing solution is added to the enzyme label plate, the added reagent is prevented from mixing into another enzyme label hole, and cross contamination is strictly prevented.
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