Related Products
  • DW111.png
DW111.png

EasySee® Super Western Blot Kit


Product Details

EasySee® Super Western Blot Kit uses enhanced lumino-based chemiluminescence for the detection of horseradish peroxidase (HRP) conjugated antibodies and the corresponding antigens. Compared with EasySee® Western Blot Kit, it is more suitable for the detection of low-abundance proteins.


Highlights

• High sensitivity, enabling the detection of picogram amounts of samples.

• Easy to use, High stability.


Storage
at 2-8℃ protected from light for two years

Shipping
ice bag (4℃) 


Product Contents


ComponentDW111-01DW111-02
Super WB Solution A 50 ml100 ml
Super WB Solution B 50 ml100 ml


Experimental Data

Western Blot Experimental Protocol


I.Experiment Materials and Reagents

1.PBS (FG701-01);

2.6× Protein Loading Buffer (DL101-02);

3.Blue Plus® IV Protein Marker (10-180 kDa) (DM131);

4.ProteinEle® Precast Tris-Glycine Gel (DG101);

5.Primary Antibodies;

6.Secondary Antibodies;

7.293T Cell Lysis Buffer (Sample);

8.Tris-Glycine Electrophoresis Buffer (Refer to TransGen Product Catalog);

9.Transfer Solution;

10.Filter Paper;

11.Methanol;

12.TBST;

13.5% Skimmed Milk;

14.Ponceau S Staining Solution (optional);

15.PVDF Membrane


II.Experimental Procedures

1.Sample Preparation

(1)Loading Volume Calculation: Taking the preparation of a 10μg sample as an example, if the sample concentration is 2μg/μl, the sample loading volume is 10μg/2μg/μ=5μl;

(2)Since the sample: 6×Loading Buffer=5:1, the volume of 6×Loading Buffer is 5μl/5=1μl;

(3)Therefore, the final loading volume is 5μl+1μ=6μl.

2.SDS-PAGE Electrophoresis

(1)Take out the precast gel, and tear off the sealing strip at the bottom of the gel. Fix the gel  in the electrophoresis chamber;

(2)Add Tris-Glycine Electrophoresis Buffer until it reaches the top of the comb well. Gently pull out the comb with both hands to prepare for sample loading;

(3)Aspirate 5μl of Blue Plus® IV Protein Marker, and add it to the loading well of the precast gel and add the quantified 293T Cell Lysis Buffer to the loading well in a gradient of 500ng, 1µg, 5µg, 10µg and 20µg. (The specific loading volume is determined according to the experimental requirements);

(4)Plug in the power supply, and set a constant voltage of 200V. Run electrophoresis for about 50 minutes, and stop electrophoresis when the bromophenol blue migrates to about 0.5cm from the bottom of the precast gel or 1/3 from the bottom of the precast gel;

(5)After electrophoresis, carefully remove the precast gel. Use the gel knife that comes with the precast gel, and carefully pry the cassette’s plate along the gaps on its left and right sides, and take out the gel. The gel can be immersed in a pre-cooled transfer solution for later use.


III.Membrane Transfer

1.Cut the PVDF membrane and 6 sheets of 3M filter paper into a size of 5.5×8.5cm 10min before the end of electrophoresis. (For this operation, it should be noted that gloves should be worn, and the gloves should be clean to avoid contaminating the PVDF membrane);

2.Pretreatment of PVDF membrane: Use tweezers to hold one end of the PVDF membrane and carefully place it into about 10 ml of methanol (Depending on the size of the incubator, if the amount of liquid is too small, the amount can be appropriately increased). Soak it until the PVDF membrane becomes translucent (about 20sec), then transfer to the pre-cooled transfer solution. Equilibrate for about 1min. And soak the filter paper at the same time for later use.

3.Lay the sponge pad, 3 layers of filter paper, gel, PVDF membrane, 3 layers of filter paper and sponge pad on the negative side of the wet transfer clip sequentially. Remove the air bubbles and clamp the clips tightly;

4.Pour the remaining transfer solution into the wet transfer tank, and cover it with  the lid. Place the wet transfer tank in an ice-water bath or refrigerator. Connect the wet transfer film transfer instrument, and set the current and transfer time. (The transfer time can be set according to the size of the target protein. The length of the transfer time is proportional to the molecular weight of the protein. The transfer conditions for this experiment are constant current 200mA, 90min)

5.After transferring the membrane, gently peel off the upper filter paper and gel with tweezers, and observe whether the marker is successfully transferred to the membrane.

6.The PVDF membrane can also be placed in Ponceau S Staining Solution for about 10 minutes, and then gently rinsed with double distilled water until clear bands are visible on the PVDF membrane. The dyed PVDF membrane can be directly blocked without other processing.


IV.Blocked

1.Use sufficient freshly prepared 5% skim milk (using TBST for preparation, put it on a magnetic stirrer to fully dissolve until there is no particles). Pour about 20ml to submerge the PVDF membrane. (Depending on the size of the antibody incubation box, if the amount of liquid is too small, the amount can be appropriately increased). Place on an inversion shaker and block at room temperature for 1-2h.


V.Antibody hybridization

1.Primary antibody hybridization: Select the appropriate hybridization time and antibody dilution ratio according to different primary antibodies (depending on the size of the antibody incubation box, it is generally appropriate to submerge the PVDF membrane. If the amount of liquid is too small, the amount can be appropriately increased)

2.Wash the membrane: After the primary antibody reaction is over (this step is optional, whether to recover the primary antibody can be determined according to the specific experimental needs. The primary antibody can be recovered and placed at 4°C, with NaNg at a final concentration of 0.1% added to prolong the storage time of the primary antibody). Use 15ml ~20ml TBST to wash off the unbound primary antibody for 4 times, 8 min for each time (Depending on the size of the antibody incubation box, it is generally appropriate to cover the PVDF membrane. If the amount of liquid is too small, the amount can be appropriately increased) .

3.Secondary antibody hybridization: Select the appropriate hybridization time and antibody dilution ratio according to different primary antibodies (depending on the size of the antibody incubation box, if the amount of liquid is too small, the amount can be appropriately increased). Place on a horizontal shaker, and incubate at room temperature for 1h

4.Wash the membrane: After the secondary antibody reaction, use 15ml~20ml TBST to wash off the unbound secondary antibody, for 4 times, 8min for each time (depending on the size of the antibody incubation box, it is generally appropriate to cover the PVDF membrane, if the amount of liquid is too small, the amount can be appropriately increased) .


VI.Visualization

1.Preparation of luminescent working solution: Mix Super WB Solution A and Super WB Solution B in equal proportions (for example, mix 1ml Super WB Solution A and 1ml Super WB Solution B in equal proportions). It is noted that  to use freshly prepared working solution

2.Use tweezers to remove the PVDF membrane from the TBST and drain the remaining liquid on the surface. But be careful not to allow the PVDF membrane to dry completely

3.Immerse the PVDF membrane completely in the chemiluminescent working solution (0.125ml chemiluminescent working solution/cm2 PVDF membrane), and incubate for 1min. Then  immediately prepare for pressing (Extending the incubation time does not increase the sensitivity and band brightness, and may also cause the appearance of non-specific bands)

CopyRight 2022 TransGen Biotech Co., Ltd. All Rights Reserved          京ICP备07502849号-1       京公网安备 11010802016705号
Message