Transfer Buffer - InstantBlot (ab270047)

Transfer Buffer - InstantBlot (ab270047)  is designed to deliver a Western blot transfer procedure in as little as ten minutes (where less than 100 kDa), without the excessive heat generation that may denature proteins.

This product is manufactured by Expedeon, an Abcam company. It was previously called InstantBlot Transfer Buffer 500ml 10X. Expedeon product code NXB87500 is the same as the 500 mL size of this product.

This transfer buffer has the advantage of compatibility with any type of blotting membrane (i.e., PVDF, nitrocellulose (NC), nylon) and does not require any special electro-blotting equipment.

INSTRUCTIONS FOR USE

How to prepare 1X Transfer Buffer - InstantBlot:

• 1. Dilute the 10X stock solution of Transfer Buffer - InstantBlot so that you have enough to fill your blotting tank. (Note: You will also need a small amount of 1X Transfer Buffer - InstantBlot for soaking your transfer membrane, blotting paper, and sponge [approx. 200ml])
• E.g., to prepare 1,500 ml of 1X Transfer Buffer - InstantBlot:
• 150 ml of 10X Transfer Buffer - InstantBlot stock solution
• 300 ml of 100% methanol (MeOH)
• 1,050 ml deionized or distilled water
• Stir for 5 minutes (i.e., with a magnetic stirrer)
• 2. Soak the blotting paper in 1X Transfer Buffer – InstantBlot for 5 minutes.
• 3. Soak the transfer membrane, PVDF or NC, in 1X Transfer Buffer - InstantBlot for 5 minutes. (Note: Prior to soaking the transfer membrane in 1X Transfer Buffer - InstantBlot, it must have been previously soaked in 100% MeOH for 2 minutes).
• 4. Soak your trimmed gel in distilled or deionized water for no longer than 2 minutes (≤2 minutes). This step removes excess SDS and does not remove all ions from the gel.

Example running conditions for blotting tank

• 1. Plug the power leads into the power supply.
• 2. Set the power supply to 400mA ‘Constant’.
• 3. Turn on the power supply and record current (mA) and voltage (V).
• 4. At a constant current of 400mA, volts should start at 90.
• 5. Proceed with the transfer for 15–20 minutes*.
• 6. After the desired length of time, turn power supply OFF.

*Transfer efficiency depends on many factors (i.e., gel concentration and thickness, protein size, shape and net charge). Transfer results may vary, therefore initial method development (e.g., trial-and-error experiments) is recommended. We also advise using a constant current during the transfer procedure, therefore you will need to make any adjustments by increasing or reducing the current (mA), and not by increasing or reducing the transfer time.

• You can verify the results of your transfer within 5 minutes using our Ponceau S. solution (ab270042).
• To verify the percentage of protein transferred, place the gel on a tray and cover with Coomassie protein stain.