制备蛋白提取物

1. Prepare extracts from cultured cells or tissues with our Trident Extraction Kits.
The total number of cells per ml and the cell equivalent loaded per lane of gel should be optimized specifically for each protein and antibody.

2. Determine the protein concentration of the extract and transfer the appropriate amount of your sample to a new tube. Aliquot and freeze the stock at -20℃ or below.

3. Add Trident Sample Buffer to your sample and boil at 100℃ for 5 minutes to denature the proteins. Spin the sample briefly and load onto your SDS-PAGE gel.
Add Dithiothreitol （DTT） or β-mercaptoethanol （2-ME） to the Trident Sample Buffer before use to reduce proteins, if necessary.



电泳和转膜

1. Load 30 μg of each protein extract or 100 ng of purified protein into the wells of the SDS-PAGE gel. Load an appropriate amount of Trident Blue Prestained Protein Ladder or Trident Prestained Protein Ladder to one or more additional lanes.

2. Run the gel in 1X Trident Running Buffer for 1-2 hours at 50-100 V.
We recommend setting the electrophoresis at a lower voltage and for a longer time. This should result in clearer bands and better resolution.

3. Transfer the proteins from the gel to a nitrocellulose or methanol-rinsed PVDF membrane in 1X Trident Transfer Buffer.
Optional: Confirm successful protein transfer by Ponceau Red staining before proceeding to the next step.



封闭，抗体孵育和洗膜

1. Blocking: Incubate the blot in 3% non-fat milk / PBST or Trident Universal Protein Blocking Reagent for 30-60 minutes at RT.

2. Primary antibody incubation: Incubate the blot in 1 % non-fat milk / PBST or Trident Universal Protein Blocking Reagent containing the primary antibody at the proper dilution for two hours at RT or 4℃ overnight.

3. Washing: Wash the blot with 1X PBST for 10 minutes once and for 5 minutes twice.

4. Secondary antibody incubation: Incubate the blot in 1 % non-fat milk / PBST or Trident Universal Protein Blocking Reagent containing the HRP-conjugated secondary antibody at the proper dilution for one hour at RT.

5. Washing: Wash the blot with 1 X PBST three times, each for 10 minutes.

The Trident Universal Protein Blocking Reagent （animal serum-free） （GTX30963） does not contain any animal serum, and can be used for all IHC and ICC kits, ELISAs, and WBs.

以 ECL 系统检测信号

1. Follow the instructions of the Trident ECL plus （GTX400006） or Trident Sharp-ECL Kit （GTX14698） for detection of your signal.