Anti-ALIX antibody [3A9] (ab117600)

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Here is the WB protocol you had requested.

Western Blotting

Reagents
1. Blocking Buffer
5% non-fat dried milk dissolved in PBS.
Note: For the detection of phosphoproteins, NEVER block membranes with milk; use 5% BSA instead. Milk contains casein (itself a phosphoprotein) which will bind phospho-specific antibodies resulting in high background.
2. Washing Buffer
Blocking buffer + 0.1% Tween-20
3. Ponceau S
Ponceau S, 0.1 g
Acetic Acid, 5 ml
Distilled Water, 95 ml
Note: Ponceau S is light sensitive.
4. PBS
Sodium Chloride, 8.0g Potassium Chloride, 0.2g
Disodium Potassium Phosphate, 1.15g
Potassium Dihydrogen Phosphate, 0.2g
Distilled Water, 1 liter
5. PBST
Sodium Chloride, 8.0g
Potassium Chloride, 0.2g
Disodium Potassium Phosphate, 1.15g
Potassium Dihydrogen Phosphate, 0.2g
Tween-20, 1.0ml
Distilled Water, 1 liter
Method
Following SDS-PAGE, transfer proteins onto blotting membrane according to the manufacturer’s instructions.
Check protein transfer by staining the blot with Ponceau S for 1 minute, then completely destain the blot by washing with distilled water.
Place blot into blocking solution for 2 hours at room temperature, or overnight at 4°C.
Rinse the blot briefly with wash buffer and then add primary antibody diluted in the wash buffer (a concentration of 1-10 µg/ml is generally acceptable and recommended). Incubate for 2 hours at room temperature, or overnight at 4°C.
Wash the blot extensively in wash buffer (3 x 10 minutes) with gentle agitation.
Add appropriate enzyme-conjugated secondary antibody (e.g. ab97023 (https://www.abcam.com/Goat-polyclonal-Secondary-Antibody-to-Mouse-IgG-HL-HRP-ab97023.html) or ab97046 (https://www.abcam.com/Rabbit-polyclonal-Secondary-Antibody-to-Mouse-IgG-H-L-HRP-ab97046.html)) diluted in wash buffer and incubate for 1 hour at room temperature with gentle agitation.
Wash the membrane with gentle agitation as follows: 4 x 5 minutes in wash buffer; 3 x 5 minutes in PBST and 2 x 5 minutes in PBS.
Add appropriate enzyme substrate solution and incubate as recommended by the manufacturer to visualize protein bands.

Appropriate controls should always be carried out. It may be useful to include a sample in which no primary antibody is used at all, in order to determine any non-specific binding of the secondary reagent to the target tissue.

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