Summary of reagents used by Western Blot
Main reagents:
1. Acrylamide and N, N'-methylenebisacrylamide should be prepared with warm deionized water (to facilitate dissolution of bisacrylamide) containing 29% (w / v) acrylamide and 1% (w / v) N, N'-methylenebisacrylamide stock solution acrylamide 29g, N, N-methylenebisacrylamide 1g, add H2O to 100ml. ) Store in a brown bottle and store at 4 ° C protected from light. Strictly verify that the PH must not exceed 7.0, because the deamination reaction can occur photocatalytic or alkaline catalyzed. The period of use shall not exceed two months, and must be reconstituted every few months. If there is precipitation, it can be filtered.
2. Sodium dodecyl sulfate SDS solution: 10% (w / v) 0.1g SDS, 1ml H2O deionized water, store at room temperature.
3. Separation gel buffer: 1.5mmol / LTris-HCL (pH8.8): 18.15g Tris and 48ml 1mol / L HCL are mixed, diluted with water to a final volume of 100ml. Store at 40C after filtration.
4. Concentrated gel buffer: 0.5mmol / LTris-HCL (pH6.8): 6.05g Tris is dissolved in 40ml H2O, adjusted to pH 6.8 with about 48ml 1mol / L HCL and diluted with water to 100ml final volume. Store at 40C after filtration. These two buffers must be prepared with Tris base, and then adjust the PH value with HCL instead of Tris.CL.
5. The original TEMED solution N, N, N'N 'tetramethylethylenediamine catalyzes the formation of free radicals by ammonium persulfate to accelerate the polymerization of two acrylamides. When the pH is too low, the polymerization reaction is suppressed. 10% (w / v) ammonium persulfate solution. Provides free radicals necessary for the polymerization of two acrylamides. Prepare a few ml of deionized water before use.
6. SDS-PAGE loading buffer: pH6.8 0.5mol / L Tris buffer 8ml, glycerol 6.4ml, 10% SDS 12.8ml, mercaptoethanol 3.2ml, 0.05% bromophenol blue 1.6ml, H2O 32ml and mix well . Mix with protein samples at a ratio of 1: 1 or 1: 2. After boiling in boiling water for 3 minutes, mix well before loading, generally 20-25ul, total protein amount 100μg.
7. Tris-glycine electrophoresis buffer: 30.3g Tris, 188g glycine, 10g SDS, dissolved in 1000ml with distilled water to obtain 0.25mol / L Tris-1.92mol / L glycine electrode buffer. Dilute 10 times before use.
8. Transfer buffer: To prepare 1 L of transfer buffer, weigh 2.9 g of glycine, 5.8 g of Tris base, and 0.37 g of SDS, add 200 ml of methanol, and add water to a total of 1 L.
9. Ponceau Stain Solution: Ponceau S 2g Trichloroacetic acid 30g Sulfosalicylic acid 30g Add water to 100ml. When the above stock solution is diluted 10 times, it becomes Ponceau S solution. It should be discarded after use.
10. Skimmed milk powder 5% (w / v).
11.NaN3 0.02% sodium azide (toxic, handled with gloves), dissolved in phosphate buffered saline (PBS).
12. Tris buffered salt solution (TBS): 20mmol / LTris / HCL (pH7.5), 500mmol / LnaCl.
13. Tween20 (15) mouse anti-human-MMP-9 (16) mouse anti-human-TIMP-1.
14. Secondary antibody labeled with peroxidase.
15. NBT (dissolved in 70% dimethylformamide, 75mg / ml).
16. BCIP (dissolved in 100% dimethylformamide, 50mg / ml).
17.100 mmol / LTris-HCL (pH9.5).
18. 100 mmol / L NaCl.
19.50mmol / LTris-HCL (pH7.5), 5mmol / L EDTA.
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