Western Blot (Immune Imprinting) Common Sense Questions and Answers

(Shanghai Lianshuo Biotechnology Co., Ltd., one of the main suppliers of domestic immunology products)

1. What is western blot?
Answer: WB is also known as immunoblot. It is the process of transferring biological macromolecular substances to the solid carrier through different channels.

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

3. What are the specific steps of western blot?
Answer: 1. Sample preparation (taking the total protein of animal tissue as an example),
1) After washing the tissue, add 3 volumes of PBS and grind at 0 ℃.
2) Add 5 × STOP buffer
3) 180W, 6mins, 0 ℃ ultrasonic breaking
4) Centrifuge at 5000rpm, 5mins, take the supernatant.
5) Add REB (0.5ml add 0.5ml), bromophenol blue (9.5ml add 0.5ml)
6) Boiling, 10min
7) Separately pack, store at -20 ℃, take out when used, and dissolve and load directly.

two. Electrophoresis (SDS-PAGE)
It is recommended to detect antigen 10-30kd, use 15% glue; 30-100kd, use 10% glue; 100-200kd, use 7.5
1) One piece A. Separating glue. Unit: ml. Total: 8ml
7.5% 10% 15%
2 × Sep. Buffer 4 4 4
30% Gel.sol 2.0 2.7 4
ddH2O 1.9 1.2 0
TEMED 8ul 8ul 8ul
10% APS 80ul 80ul 80ul
B. Concentrated gel. Unit: ml. Total: 3.5ml
2 × Stacking. Buffer 1.7
30% Gel.sol 0.35
ddH2O 1.4
10% APS 50ul

2) Spotting. The amount of loaded protein should not exceed 30ug / mm2. (The loading surface is: if your glue groove is 5mm × 1mm, the loading surface is: 1mm × 5mm = 5mm2)

3) Electrophoresis. Start with a voltage of 80V, but when bromophenol blue reaches the separation gel, use a voltage of 100-120V.

4) Transfer (one piece of glue).
A. Semi-dry method.
a. Take six sheets of filter paper (Bio-Rad, 1mm), three sheets as the upper filter paper, and three sheets as the lower layer. among them,
The upper filter paper must be relatively clean and free of pollution. Soak in Transfer buffer for at least five minutes before use.
b. Prepare nitrocellulose film, soak it in methanol for 1-3min. After pouring, use Transfer
buffer soak.
c. Make sandwiches. Put three pieces of lower filter paper on the anode, re-soaked membrane, glue, and then put on the upper layer of filter paper in the three experimental method mutual aid area-protein area, and finally put three pieces of upper filter paper on, and cover the cathode. Note that there must be no air bubbles between each layer. Use a pencil to draw the edge of the glue. Mark up and down.
d. Transfer, 0.8mA / cm2-3mA / cm2. Experience is: 100kd-200kd, use 2mA / cm2, 2hrs;
30-100kd use 1.0-2.0mA / cm2, 40mins-1.5h; 10kd-30kd% use 0.5-0.8mA / cm2,
B. Wet method.
a. Take four filter papers, two as the upper filter paper and two as the lower filter paper. Among them, the upper filter paper must be relatively clean and free of pollution.
b. Prepare nitrocellulose film, soak it with methanol for 1-3mins. After pouring, use Transfer
buffer soak.
c. Make sandwiches.
e. Transfer. Antigen ≥100kd, first transfer 28V for 1h, then 84V transfer 14-16h, ≤100kd, then
63V transfer 4-16h.

Three blocking: 5% milk is blocked overnight at 4 ℃, or RT 1hr.

4. The first step reaction: 5% milk or 2% BSA diluted antibody (called the primary antibody), the dilution ratio according to the antibody instructions, incubated at room temperature for 1hr

Five washing: TBST washing 3-5 times for 5 mins.

6. Second step reaction: Incubate the membrane with 2 antibodies diluted in 5% milk or 2% BSA at room temperature for 1 hour.

Seven washing: TBST washing 3-5 times for 5 mins.
Eight color rendering:

4. What is the formula of the buffer needed for western blot?
Answer: 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
Experimental Method Mutual Assistance Area-Protein Area
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, add 0.5ml β-ME and 0.5ml bromophenol blue to make sample buffer.

5. What are the common problems of western blot?
1) Why is there no target protein in my cell extract?
Answer: There are many reasons. 1 Your cell does not express this protein. Change to another cell. 2 The protein in your cell is degraded. You must add PMST to inhibit protease activity. 3 Your antibody cannot recognize the target. For protein, look at the instructions to see if there is a problem.
2) Some of my cell extracts are precipitated and some are clear, why?
Answer: 1 There may be precipitation because your protein is not completely denatured. You can increase the SDS concentration appropriately and increase the boiling time of the sample. 2 It does not rule out that your antigen concentration is too high. Then add an appropriate amount of sample buffer.
3) The molecular weight of the protein I made is very small (10KD), how do I make WB?
Answer: You can choose PSQ membrane and shorten the transfer time. You can also stack the two films together and transfer them. Just follow the steps.
4) My goal belt is weak, how can I strengthen it?
Answer: You can increase the amount of antigen loaded. This is the most important. At the same time, the dilution ratio of primary antibody can also be reduced.
Experimental Method Mutual Assistance Area-Protein Area
5) What is the solution to the dirty background of the film?
Answer: Reduce the amount of antigen loading, 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 it. Why?
Answer: The concentration of your primary antibody is high, and the catalytic activity of HRP on the secondary antibody is too strong. At the same time, your chromogenic substrate is at a critical point. The reaction time is not long. The surrounding substrate is catalyzed and a blank is formed. Reduce the primary and secondary antibody concentrations, or replace with new substrates.
7) My film is blank, what's wrong?
Answer: If you can eliminate the following problems, most of the problems will occur in the preparation of primary antibodies and antigens.
1 The HRP activity of the secondary antibody is too strong and consumes the substrate; 2 H2O2 in the ECL substrate is unstable and inactive; 3 ECL
The substrate did not cover the corresponding position; 4 the secondary antibody was inactivated.
8) I asked the developer to develop the film for 1 minute, and after 5 minutes, the film is dark. What is the reason?
Answer: 1 may be caused by a red light, you can operate in a completely dark situation. See if there is improvement.
The film was originally exposed. 2 The development time was too long.
9) Is DAB or ECL good?
Answer: DAB is toxic, but more sensitive, it is the most sensitive substrate for HRP;
The ECL result is easy to control, but the sensitivity is a bit worse when catalyzed, but if it reaches the threshold, it is particularly sensitive and can detect pg-level antigen. It depends on your experiment.
10) The molecular weight of the antigen is twice that on the data sheet, what is going on?
Answer: The antigen has formed a dimer. Increase the amount of mercaptoethanol, prolong the boiling denaturation time, can open the dimer. It can also be opened by adding urea to 8M in the upper glue.
11) Does semi-dry transfer require the same size of membrane, filter paper and glue?
Because the size of the glue is not necessarily regular, the membrane and filter paper will be larger, will it short circuit?
What is a short circuit? How to control and discover short circuits?
Answer: generally refer to membrane> = filter paper> glue, just do,
Just look at the voltage before and during the transfer. The normal semi-dry is slowly becoming higher. At the end, it is generally 1.5-3 times the beginning. Generally, the selected buffer and filter paper will not short-circuit.
12) Marker added 5ul according to the instructions during electrophoresis, but it is invisible during electrophoresis, is it inactivated?
Answer: Just add 20ul.
13) The protein can not be transferred to the membrane, but it is on the glue, and the Marker is transferred. Why?
Answer: It may be that the sample concentration is too low, and the transfer time is not enough.
14) I have a small amount of primary antibody, and I don't know what the optimal dilution ratio of primary antibody is. What should I do?
Answer: After confirming that there is no pollution during the experiment, the antibody-containing dilution is recovered and stored at -70 ℃. It is best to increase the concentration for the first time, so that if the result is not good, you can try to dilute it.

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