Three FAQs in Western Blot

Western Blot has become a routine means of detecting proteins in the laboratory. However, it is still challenging to get a picture with clear lines, no banding, and a clean background. When we do protein electrophoresis and Western Blot, most of them use Bio-Rad's instruments and abcam's antibodies, so the technical experts in Bio-Rad protein can indeed be called experts because many people ask them every day. . They organized the most common questions into "Western Blotting Troubleshooter." We have selected some of the sections, hoping to save you some time searching for information.

Brands

Antibody name

Item number

specification

Market price

Sale price

Abcam

Anti-Chk2 (phospho T387)

Ab55319

100 Î¼g

3996.00

3196.00

Abcam

Anti-Chk1 [EPR3952]

Ab133277

100 Î¼l

4175.00

3340.00

Abcam

Anti-CDKN2A

Ab189034

50 Î¼g

3996.00

3196.00

Abcam

Anti-ATR Antibody [2B5]

Ab4471

100 Î¼g

4255.00

3404.00

Q: My result is a blank film. Why is this?

A: There are many factors that cause this result.

The glue and film are reversed. If the position of the glue and the film is reversed during the transfer process, the protein will be transferred from the glue to the buffer and will not reach the film. For standard transfer, the gel should be near the negative electrode of the sandwich and the membrane should correspond to the positive electrode.

Low membrane efficiency The membrane efficiency is affected by many factors, including the size of the protein, the percentage of acrylamide in the gel, the strength of the electric field, the transfer time, and the pH of the buffer. In general, the larger the protein, the slower the transfer. The best way to transfer large proteins is to use high electric field strength. Small proteins may pass through the transfer film for a long time under high electric field strength. The way to avoid this problem is to transfer with 0.2 um pore size NC membrane or PVDF membrane. If the isoelectric point of the protein is close to the pH of the buffer, then this protein carries little charge and hardly moves in the electric field. If your target protein is strongly alkaline, then you can use carbonate (pH 9.9), CAPS (pH 11) and acidic buffers.

After transfer, you can determine the transfer efficiency by dyeing or a second film. To help you directly monitor transfer efficiency, Bio-Rad also offers a variety of pre-stained Markers that can be observed directly after electrophoresis and transfer. If the reagent is left for too long or the storage conditions are incorrect, the antibody will slowly degrade. If it freezes and thawing repeatedly, it will degrade rapidly. The substrate should be stored at -20Â°C.

The antibody concentration is too low or the titer is too low. The concentration of the primary antibody varies widely. The dilution of the primary antibody should be determined empirically. Too often, too many antibodies will prevent the binding of antigens to antibodies. The general principle is to dilute the serum or tissue culture supernatant 1:100-1:1000 and dilute the serum of ascites or hyperimmune animals 1:1000-1:10000. Bio-Rad's blot-level secondary antibody dilution is 1:3000.

Inactivation of sodium azide by the enzyme is an inhibitor of horseradish peroxidase. Do not use any reagent containing sodium azide in HRP-colored western blots. Sodium azide can be used in alkaline phosphatase-conjugated antibodies without side effects. In addition, tap water or water deionized with a polystyrene resin may also deactivate enzymes, using only distilled deionized water.

Washing Tween-20 (Tween-20) with Tween-20 may interfere with certain antibody-antibody interactions or wash off the target protein on the NC membrane. Although its final concentration is only 0.05%-0.1% when washed, it may still affect the binding. Therefore, Tween-20 is not used in other washing processes except for the first wash after closing, but this may cause the background to rise.

The detection system lacks sufficient sensitivity to ensure that the amount of protein loaded is within the detection system's sensitivity range:

Horseradish Peroxidase 500 pg/band
Alkaline phosphatase 100 pg/band
Enhanced alkaline phosphatase 5 pg/band
Colloidal Gold 100 pg/band
Enhanced Colloidal Gold 10 pg/band
Immun-Star Chemiluminescence 10 pg/band

Q: My western blot is always too high background. How to solve it?

A: High background may be caused by many factors: incomplete sealing, excessive color development, inadequate washing, contamination of the transfer buffer or equipment, incorrect antibody dilution, or impure antibody.