Immunoblot (WB) FAQ

1. What is a western blot?
A: WB, also known as immunoblotting, is the process of transferring biological macromolecules to solid supports by different routes.

2. What are the advantages of western blot?
A: Sensitive, up to ng level, theoretically up to pg level with Ecl color rendering. Convenient and high specificity.

3. What are the specific steps of western blot?
Answer: 1. Sample preparation (to take animal tissue total protein as an example),

1) After tissue washing, 3 times volume of PBS was added and ground at 0 °C.
2) Add 5×STOP buffer
3) 180W, 6mins, 0°C ultrasonic fracture
4) Centrifuge at 5000 rpm for 5 mins and take the supernatant.
5) Add REB (9.5ml added to 0.5ml), bromophenol blue (9.5ml added to 0.5ml)
6) Boil, 10min
7) Dispense, store at -20 °C, take it out when it is used, and dissolve it directly.
two. Electrophoresis (SDS-PAGE)
It is recommended to test the antigen 10-30kd, use 15% glue; 30-100kd, use 10% glue; 100-200kd, use 7.5% glue.
1) One piece

2) Spotting. The amount of protein to be applied should not exceed 30ug/mm2. (Load surface: if your tank is 5mm × 1mm, the load surface is: 1mm × 5mm = 5mm2),
3) Electrophoresis. Start with 80V, but when bromophenol blue to the separation gel, use 100-120V.
4) Transfer (a piece of glue).
A. Semi-dry method.
a. Take six filter papers (Bio-Rad, 1mm), three for the upper filter paper, and three for the lower layer. Among them, the upper filter paper must be relatively clean and free of pollution. Soak in the Transfer buffer for at least five minutes before use.
b. Prepare a nitrocellulose membrane and soak it in methanol for 1-3 min. After draining, soak it in Transfer buffer.
c. Make a sandwich. Three lower filter papers are laid on the anode, the film is soaked, and then the glue is applied. Then, three upper filter papers are laid, and finally three upper filter papers are laid and the cathode is covered. Note that there should be no bubbles between each layer. The edge of the glue is drawn with a pencil on the film. Mark up and down.
d. Transfer, 0.8 mA/cm 2-3 mA/cm2. Experience is: 100kd-200kd, with 2mA/cm2, 2hrs; 30-100kd with 1.0-2.0mA/cm2, 40mins-1.5h; 10kd-30kd% with 0.5- 0.8mA/cm2, 15mins-40mins.
B. Wet method.
a. Take four sheets of filter paper, two for the upper filter paper, and two for the lower layer. Among them, the upper filter paper must be relatively clean and free of pollution.
b. Prepare a nitrocellulose membrane. Soak it in methanol for 1-3mins. Drain it and then soak it in Transfer buffer.
c. Make a sandwich.
e. Transfer. The antigen is ≥100kd, first transferred at 28V for 1h, then transferred at 84V for 14-16h, ≤100kd, then 63V is transferred for 4-16h.
three. Closed: 5% milk blocked at 4 ° C overnight, or RT 1 hr.
four. The first step reaction: 5% milk or 2% BSA diluted antibody (called primary antibody), the dilution ratio according to the antibody instructions, incubation at room temperature for 1 hr,
Fives. Wash: TBST wash 5 mins 3-5 times.
six. Step 2: Incubate the membrane with 5% milk or 2% BSA diluted 2 antibody for 1 hr at room temperature.
Seven. Wash: TBST wash 5 mins 3-5 times.
Eight. Color development:

4. What is the recipe for the buffer required for western blot?
A: The main buffers are:

1) 2×Separation buffer (PH: 8.8)
0.75M TRIS.HCl 90.825g
0.2% SDS 10ml 20% SDS
Total: 1000ml

2) 30% gel Solution
0.8% kis-aciylamide 0.8g
29.2% aciylamide 29.2g
Total: 100ml

3) 10% APS: 0.1g dissolved in 1.0ml ddH2O
.
4) 2×Stacking buffer (PH: 6.8)
0.2M Tris 15.14g
0.2% SDS 1g or 5ml 20% SDS
Total: 500ml

5) 10×Running buffer (PH: 8.3)
250mM Tris 60.55g
1.9M Glycine 285.27g
1% SDS 20g or 100ml 20% SDS
Total: 2 liters

6) Semi-dry transfer buffer (PH: 8.3):
48mM Tris 5.8g
39mM Glycine 2.9g
0.037% SDS 0.37g
20% MeOH 200ml
Total: 1000ml

7) Wet transfer buffer 1 (PH: 8.3): (20-400kd)
50mM Tris 5.8g
380mM Glycine 29g
0.1% SDS 1.0g
20% MeOH 200ml
Total: 1000ml

8) Wet transfer buffer 2 (PH: 8.3) <80kd
25mM Tris 5.8g
190mM Glycine 29g
20% MeOH 200ml
Total: 1000ml

9) blocking buffer:
5% milk 5g
TBST 100ml
※: Milk powder is skimmed milk powder.

10) 10×TBS (PH: 7.6)
NaCl 80g
Tris 30g
Total: 1000ml

11) TBST (PH: 7.4)
NaCl 25mM
Tris 100Mm
Tween-20 0.2%
Total: 1000ml

12) 5×STOP buffer (PH: 6.7)
20% Glycerol 100ml
10% SDS 50g
250mM Tris 15.14g
Take 9.0ml when used, add 0.5ml β-ME and 0.5ml bromophenol blue to make sample buffer.

5. Western blot What are the common problems?

1) Why is there no target protein in my cell extract?
A: There are many reasons. 1 You don't express this protein in your cells, you can change it. 2 The protein in your cell is degraded. You must add PMST to inhibit protease activity. 3 Your antibody does not recognize the target. Protein, look at the instructions and see if there is a problem.
2) Some of my cell extracts have precipitates, some are very clear, why?
Answer: 1 There may be precipitation because your protein is not completely denatured. You can increase the SDS concentration and prolong the boiling time of the sample. 2 It is not excluded that your antigen concentration is too high. Then add the appropriate amount of sample buffer.
3) The molecular weight of the protein I made is very small (10KD). How do I do WB?
A: You can choose the PSQ film and shorten the transfer time. It is also possible to stack the two films together and transfer them. Other steps can be used.
4) My purpose is very weak, how to strengthen?
A: You can increase the amount of antigen loaded. This is the main thing. At the same time, the dilution ratio of the primary antibody can also be lowered.
5) The film background is dirty. Is there any solution?
A: Reduce the amount of antigen loaded, reduce the concentration of primary antibody, change the incubation time of primary antibody, and increase the milk concentration.
6) The target band is blank and there is a background around. Why?
A: Your primary antibody concentration is high, the HRP catalytic activity on the secondary antibody is too strong, and your chromogenic substrate is at a critical point. The reaction time is not long, and the surrounding substrate is catalyzed to form a blank. Reduce the concentration of primary and secondary antibodies, or replace with new ones.
7) My film is a blank, what is going on?
A: If you can eliminate the following problems, then most of the problems appear in the primary antibody and antigen preparation. 1 The HRP activity of the secondary antibody is too strong, and the substrate consumes light; 2 H2O2 in the ECL substrate is unstable and inactivated; 3 ECL
The substrate did not cover the corresponding position; 4 the secondary antibody was inactivated.
8) Ask me to develop the developer for 1 minute, and after 5 minutes, the film is dark, what is the reason?
Answer: 1 may be caused by a red light. It can be operated in complete darkness. See if there is any improvement in the film that was originally exposed. 2 The development time is too long.
9) Is DAB good or ECL good?
A: DAB is toxic, but sensitive. It is the most sensitive substrate for HRP. ECL results are easy to control, but the sensitivity is poor when catalyzed, but if it reaches the threshold, it is particularly sensitive and can detect pg-level antigen. It depends on the situation of your experiment.
10) The molecular weight of the antigen is twice as much as the data. What is going on?
A: The antigen forms a dimer. Increase the amount of mercaptoethanol, prolong the boiling denaturation time, and open the dimer. It is also possible to add urea to the top layer of glue to 8M.
11) Does semi-drying require the same size of membrane, filter paper and glue? Because the size of the glue is not necessarily regular, the film and filter paper will be larger, so will it be short-circuited? What is a short circuit? How to control and find a short circuit?
A: Generally refer to the film >= filter paper > glue, on the line, you can see the voltage before the transfer and transfer process, the normal semi-dry is slowly become higher, the final end is generally 1.5-3 times the beginning is normal of. Generally, the pair of Buffer and filter paper selection will not be short-circuited.
12) Marker adds 5ul according to the instructions during electrophoresis, but it is invisible in electrophoresis. Is it inactivated?
A: Add 20ul.
13) The protein is not transferred to the membrane, but it is on the gel, and Marker turns it up. Why?
A: It may be that the sample concentration is too low and the transfer time is not enough.
14) My primary antibody is less, and I don't know the optimal dilution ratio of the primary antibody. How do I do it?
A: After confirming that the experiment process is non-polluting, the antibody-containing dilution is recovered and stored at -70 °C. It is best to make the concentration higher for the first time, so if the result is not good, try to dilute again.