Does your lab have both researchers that use PVDF membranes and researchers that use nitrocellulose for infrared Western blots? Are you still spending your valuable time making homemade stripping buffer?
NewBlot™ IR Stripping Buffer to the rescue! NewBlot IR is the latest member of the LI-COR NewBlot Stripping Buffer family.
All NewBlot buffers can be used for stripping and reprobing infrared fluorescent Western blots, and there is a formulation that works for whichever membrane type (or types) you use.
What makes NewBlot IR unique?
- It strips both membrane types so you won’t need to buy separate stripping buffers for nitrocellulose and PVDF.
- It is the most affordable, saving you money to spend on other items for your lab.
This robust reagent removes primary and secondary antibodies while maintaining target antigen integrity for efficient reprobing and does not require hazardous shipping, unlike many other stripping buffers.
Note: NewBlot IR Stripping Buffer is not recommended for loading amounts over 30 µg.
Below are data showing that you can indeed strip and reprobe three times and still detect your proteins of interest.
Figure 1. Strip and reprobe nitrocellulose or PVDF membranes effectively with NewBlot IR Stripping Buffer. EGFR and phospho-ERK levels were compared in EGF-stimulated (+) and non-stimulated (-) A431 lysate (1 µg total protein). The membrane was probed with mouse anti-EGFR, mouse anti-pERK1/2, and β-tubulin rabbit polyclonal (LI-COR P/N 926-42211), then detected with IRDye 800CW Goat anti-Mouse (LI-COR P/N 926-32210) and IRDye 680RD Goat anti-Rabbit (LI-COR P/N 926-68071). The blot was scanned with an Odyssey® CLx Imaging System (Original blot). The blot was then stripped with NewBlot IR Stripping Buffer (LI-COR P/N 928-40028), and scanned again (Strip #1). The blot was detected with the same primary and secondary antibodies and scanned again (Reprobe #1). The process was repeated 2 more times (Strip and Reprobe #2-3). Image display settings for all stripped and reprobed images are identical to the original image.
To make sure you are successful when stripping and reprobing, here are some factors that can affect stripping efficiency:
- Amount of time the blot is in the stripping buffer
- Sample type and preparation
- Blot handling conditions
- Buffer concentration and temperature used for stripping
Save your valuable time and money! Order NewBlot IR Stripping Buffer today for your infrared fluorescent Western blot stripping and reprobing needs.
NewBlot Western Blot Stripping Buffers are specially formulated for use with IRDye® infrared dyes (680RD, 680LT, and 800CW only) and the Odyssey® Infrared Imaging Systems to help you save time and money on recreating samples. NewBlot Stripping Buffer allows you to reuse the same blot by stripping and reprobing up to two fluorescent antibodies simultaneously.
So, you may ask, what’s so great about NewBlot Stripping Buffer?
- Effectively removes antibodies, yet gentle enough to retain immobilized proteins
- Strips blots at room temperature in 20 minutes or less without an unpleasant odor
- Allows you to reuse the same blot up to 3X! (see the data below!)
- Offers qualitative analysis after stripping
In the example below, beta tubulin and ERK2 were run on a gel and transferred to Immobilon®-FL PVDF membrane. They were probed with primary antibodies rabbit anti-beta-tubulin and mouse anti-ERK2 and then with IRDye 680 Goat anti-Rabbit (red) and IRDye 800CW Goat anti-Mouse (green), respectively. NewBlot PVDF Stripping Buffer was used to strip the blot, which was then reprobed with the fluorescent secondary antibodies. This was repeated 2 more times. As you can see from the series of images, there is very little apparent loss of signal in either channel in the third blot as compared to the original blot.
NewBlot is available in two ‘flavors’: one for stripping nitrocellulose membranes and the other for stripping PVDF membranes.
Note: On August 25, 2014, we launched two new Western blot stripping buffers: NewBlot™ IR Stripping Buffer for infrared Western blots on either PVDF OR nitrocellulose membranes; and, WesternSure® ECL Stripping Buffer for chemiluminescent Western blot stripping and reprobing. BOTH do not require hazardous shipping charges, unlike many other Western stripping buffers.