This is a lab book, but the experimental results of the experimenter have problems:
Light color, low sensitivity
.
Through this lab book, our company gives a professional analysis:
l
Keshun technical answer:
The kit is not fully balanced
l
Solution
: Remove the kit from the 2 to 8 ° C refrigerator, open the lid, and equilibrate for at least 20 minutes at room temperature to ensure that all reagents are equilibrated to room temperature (about 25 ° C)
l
Keshun technical answer:
The ELISA plate is excessively dried when it is stored or washed.
l
Solution:
Prevent the board from being too dry
Experimental principle:
The kit uses a double antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA). Pre-coated
Human C polypeptide
(
CP
The coated antibody is coated in the microwell, and the sample, the standard, and the HRP-labeled detection antibody are sequentially added, and the cells are washed and thoroughly washed. Using the substrate TMB to develop color, TMB is converted to blue under the catalysis of peroxidase and converted to the final yellow color by the action of an acid. The depth of the color and the sample
Human C polypeptide
(
CP
) is positively correlated. The absorbance (OD value) was measured at a wavelength of 450 nm using a microplate reader to calculate the sample concentration.
Sample processing and requirements
1.
serum
:
Whole blood samples collected in serum separation tubes were allowed to stand at room temperature for 2 hours or 4
°C
Overnight, then centrifuge at 1000 × g for 20 minutes, take the supernatant, or put the supernatant in -20
°C
Or -80
°C
Save, but avoid freezing and thawing repeatedly.
2.
plasma
:
Collect specimens with EDTA or heparin as anticoagulant and place the specimens at 2-8 within 30 minutes of collection.
°C
Centrifuge at 1000 × g for 15 minutes, take the supernatant to detect, or place the supernatant at -20
°C
Or -80
°C
Save, but avoid freezing and thawing repeatedly.
3. Tissue homogenization:
The tissue was washed with pre-cooled PBS (0.01 M, pH = 7.4) to remove residual blood (the lysed red blood cells in the homogenate affected the measurement), and the tissue was cut after weighing. The shredded tissue and the corresponding volume of PBS (generally in a weight ratio of 1:9, such as 1g of tissue sample corresponding to 9mL of PBS, the specific volume can be adjusted according to the needs of the experiment, and record. Recommended to add in PBS The protease inhibitor) was added to a glass homogenizer and thoroughly ground on ice. To further lyse tissue cells, the homogenate can be sonicated or repeatedly thawed. Finally, the homogenate was centrifuged at 5000×g for 5-10 minutes, and the supernatant was taken for detection.
4. Cell culture supernatant or other biological specimens:
Centrifuge at 1000 × g for 20 minutes, take the supernatant to test, or store the supernatant at -20 ° C or -80 ° C, but avoid repeated freezing and thawing.
Note: Specimen hemolysis will affect the final test results, so hemolysis specimens should not be tested.
Required reagents and equipment
1.
Microplate reader (450nm)
2.
High-precision sampler and tip: 0.5-10uL, 2-20uL, 20-200uL, 200-1000uL
3.
37 ° C incubator
4.
Distilled or deionized water
Kit composition
Prepare
Note
:
1.
The standard concentration is: 80
, 40, 20, 10, 5, 2.5
Ng/ml
2. After a large number of normal specimen tests, the normal concentration values ​​of the specimens are within the detection range provided by the kit, and 50 is taken directly during the experiment.
μL
The sample can be loaded. When some sample values ​​exceed the maximum standard concentration, the sample may be diluted with the sample dilution before the experiment.
Reagent preparation
The kit should be taken out of the refrigerated environment and should be balanced at room temperature before use.
2
0× Washing buffer dilution: Distilled water was diluted 1:20, ie 1 part of 20× washing buffer plus 19 parts of distilled water.
Steps
1.
Balance from room temperature
15
Min
The required slats were taken out from the back aluminum foil bag, and the remaining slats were sealed back to 4 ° C with a ziplock bag.
2.
Set standard and sample wells, standard wells with different concentrations of standard 50μL;
3.
Sample hole
in
plus
Enter
Sample to be tested
5
0 μL; blank holes are not added.
4.
In addition to the blank wells, 100 μL of horseradish peroxidase (HRP)-labeled detection antibody was added to each well of the standard well and the sample well, and the reaction well was sealed with a sealing plate, and incubated at 37 ° C in a water bath or incubator for 60 min.
5.
Discard the liquid, pat dry on absorbent paper, fill each well with washing solution
(350
μL
)
, let stand for 1 min, remove the washing liquid, pat dry on the absorbent paper,
Repeated washing
8
Times
(It can also be washed by a washer.)
6.
50 μL of each of the substrates A and B was added to each well, and incubated at 37 ° C for 15 min in the dark.
7.
50 μL of the stop solution was added to each well, and the OD value of each well was measured at a wavelength of 450 nm within 15 min.
Experimental result calculation
Take
OD value of the tested standard
For the abscissa,
Standard concentration
Value is the ordinate, on the coordinate paper
Or draw with related software
standard curve line
And get
Linear regression equation
,
Substituting the OD value of the sample into the equation to calculate the sample
of
concentration
.
Company quality product recommendation:
people
FMS-like tyrosine kinase 3 (Flt3) elisa kit
people
FMS-like tyrosine kinase 3 ligand (Flt-3L) ELISA kit
people
FOS-like antigen 2 (FOSL2) ELISA kit
people
F actin (Factin) ELISA kit
people
G1/S specific cyclin E1 (CCNE1) ELISA kit
people
GATA Binding Protein 2 (GATA2) ELISA Kit
people
GCSFReceptor (CD114) ELISA Kit
people
GDP Dissociation Inhibitor 2 (GdI2) ELISA Kit
people
Gremlin1 Protein (GREM1) ELISA Kit
people
GTPase NRas (NRAS) ELISA Kit
people
GTPase Activated Protein (GAP) ELISA Kit
people
G-filled FHA domain angiogenic factor 1 (AGGF1) ELISA kit
people
G protein coupled receptor 44 (GPR44/CRTH2/DL1R) ELISA kit
people
G protein coupled estrogen receptor 1 (GPR30) ELISA kit
A membrane keyboard is a keyboard whose keys are embedded in a single platform without separate moving parts. Usually, the keyboard is designed with separate keys, and each key is an independent moving part.
But the membrane keyboard has taken a step in a different direction with a pressure pad with key outlines and symbols printed on it.
Membrane Keypads,Waterproof Membrane Keypad,Membrane Keypad For Telecommunication Equipment,Mechanical Membrane Keypad
KEDA MEMBRANE TECHNOLOGY CO., LTD , https://www.kedamembrane.com