NewBlot PVDF 5X Stripping Buffer

Transcription

1 NewBlot PVDF 5X Strippig Buffer Developed for: Odyssey Family of Imagers Please refer to your maual to cofirm that this protocol is appropriate for the applicatios compatible with your model of Odyssey Imager. Part Number: Pack Size: 100 ml Storage: Room temperature Published February The most recet versio of this pack isert is posted at

2 NewBlot PVDF 5X Strippig Buffer Page 2 Table of Cotets Page I. Strippig Guidelies II. Procedure III. Optimizatio Guide IV. Frequetly Asked Questios

3 NewBlot PVDF 5X Strippig Buffer Page 3 Reagets Provided NewBlot PVDF 5X Strippig Buffer, 100 ml (sufficiet for up to 3000 cm 2 or up to fifty 7 x 8.5 cm Millipore Immobilo -FL PVDF membraes) Reagets Needed 1X PBS wash buffer Ultrapure water Icubatio Box - Large (LI-COR, P/N ) or equivalet Completed Wester blot to be stripped (wet) I. Strippig Guidelies Familiarize yourself with the etire Procedure, Optimizatio Guide, ad Frequetly Asked Questios before proceedig. Esure that you have the ecessary supplies ad reagets for strippig the membrae. Always prepare fresh workig solutio for each blot to be stripped. The workig solutio of strippig buffer should oly be reused whe a blot requires a exteded amout of time to remove ligerig fluorescet sigal (after the iitial strippig step). NewBlot PVDF Strippig Buffer is provided as a 5X cocetrated solutio. Do ot allow the membrae to become dry at ay poit durig the Wester assay or the strippig process. If you must leave the membrae uatteded betwee steps, store it i 1X PBS buffer. NewBlot PVDF Strippig Buffer has bee optimized for use with LI-COR IRDye atibody cojugates ad Millipore Immobilo-FL PVDF membrae. Wester blots probed with IRDye 800CW, IRDye 680LT, or IRDye 680RD cojugates ca be effectively stripped usig NewBlot Strippig Buffer. NewBlot Strippig Buffer should ot be used with IRDye 700DX cojugates. NewBlot Strippig Buffer may perform well with PVDF membraes or atibody-dye cojugates from other maufacturers, but LI-COR either guaratees or supports such use.

4 NewBlot PVDF 5X Strippig Buffer Page 4 II. Procedure 1. Prepare a 1X workig solutio by mixig oe part NewBlot PVDF 5X Strippig Buffer with four parts water. The large LI-COR icubatio box (11.75 x 8.9 cm, P/N ) with a 7 x 8.5 cm membrae requires a miimum volume of 20 ml. Optimizatio of the workig solutio cocetratio may be required. 2. Trasfer workig solutio of NewBlot PVDF Strippig Buffer to a clea icubatio cotaier. 3. Place blot to be stripped ito the icubatio cotaier. Make sure the blot is fully submerged ad ca move freely withi the cotaier. Place cotaier oto shaker/rotator ad allow to shake briskly (70-80 rpm) for 20 miutes at room temperature. 4. Immediately rise blot by removig from strippig buffer ad placig ito a fresh cotaier of 1X PBS; use eough PBS to completely submerge the blot. Repeat this step two more times i successio usig fresh 1X PBS each time (a total of 3 rises). NOTE: Allow blot to remai i 1X PBS after the last rise util further processig. Do ot allow the blot to dry. 5. Image the blot o a istrumet from the Odyssey family of imagers to esure complete removal of sample fluorescece. 6. If sample fluorescece remais, refer to Optimizatio Guide; otherwise, proceed to reprobe the blot with the desired primary ad subsequet secodary atibodies.

5 NewBlot PVDF 5X Strippig Buffer Page 5 III. Optimizatio Guide The three mai factors which affect strippig efficacy o PVDF membraes are time, temperature, ad iclusio of SDS deterget. If there remais sigificat sigal o the blot after completig the previous Procedure, cotiue to strip the blot as follows, util the desired results are achieved: 1. Place the blot back ito the 1X workig solutio of NewBlot PVDF Strippig Buffer. Place cotaier oto shaker/rotator ad allow to shake briskly for 10 miutes (room temperature). 2. Immediately rise blot by removig from strippig buffer ad placig ito a fresh cotaier of 1X PBS. Repeat rise two more times i successio usig fresh 1X PBS each time. 3. Image the blot o a istrumet from the Odyssey family of imagers to esure complete atibody removal. 4. If fluorescet sigal remais, add Sodium Dodecyl Sulfate (SDS) to the Strippig Buffer cotaier, to a fial cocetratio of 0.5% (v/v), ad mix. Place blot ito the 1X Buffer + SDS solutio ad shake briskly for 5 miutes (room temperature). 5. Immediately rise by removig blot from strippig buffer ad placig ito a fresh cotaier of 1X PBS. Repeat rise two more times i successio usig fresh 1X PBS each time. 6. Image the blot o a istrumet from the Odyssey family of imagers to esure complete atibody removal. 7. If fluorescet sigal remais at this poit, icubatio time ca be exteded i icremets of 5 or 10 mi, util desired results are achieved. OPTIONAL: Icubatio i Strippig Buffer ca be carried out at 37 C usig a water bath or warm-air icubator. This should oly be attempted if the above optimizatio steps are usuccessful, sice heatig durig the strippig process will sigificatly reduce the ability to successfully reprobe the blot. Do ot allow the solutio or the membrae to overheat. Icreasig the strippig time ad temperature, ad addig SDS to the strippig solutio will progressively reduce the ability to successfully reprobe the blot due to the icreased likelihood of damage to the target atige. Followig the precedig optimizatio guidelies i step-wise fashio will help to miimize this damage, while improvig the chaces of successful fluorescet sigal removal. I some cases, fluorescet sigal caot be removed completely, eve uder the most striget strippig coditios. This is due to several factors, icludig sample load amout, atibody affiity/avidity, ad target protei abudace. Oe factor which ca greatly reduce the ability to successfully strip ad reprobe a blot is whether or ot the blot was allowed to dry at all durig icubatio, washig, scaig, ad strippig steps. Make certai that the blot is kept moist at all times.

6 NewBlot PVDF 5X Strippig Buffer Page 6 The example data show i the followig figures illustrate the relative strip-ad-reprobe effectiveess of icreasig time ad additio of SDS. The sample ad protei targets, i this case, were ot sufficietly stripped usig the stadard strippig Procedure, so optimizatio was required. A IRDye 680LT Goat ati-rabbit Percet Sigal Remaiig After Strippig B IRDye 680LT Goat ati-mouse Percet Sigal Remaiig After Strippig (=3) 1X NewBlot 20 mi 1X NewBlot 60 mi 1X NewBlot mi with SDS 1X NewBlot 20 + SDS 20 mi (=3) 1X NewBlot 20 mi 1X NewBlot 60 mi 1X New wblot mi with SDS 1X NewBlot 20 + SDS 20 mi Figure 1. Relative strippig effectiveess o (A) Acti ad IRDye 680LT Goat ati-rabbit atibodies, ad (B) EGFR ad IRDye 800CW Goat ati-mouse atibodies with differet Strippig Buffer coditios: (from left) 1X NewBlot for 20 mi (stadard coditios), 1X NewBlot for 60 mi; 1X NewBlot for 20 mi, followed by a additioal 5 mi icubatio after the additio of SDS; 1X NewBlot + SDS for 20 mi. Figure 2. Images showig a example of Wester blot strippig optimizatio with NewBlot PVDF Strippig Buffer. (A) Iitial Wester blot, showig EGFR detected with IRDye 800CW Goat ati- Mouse ad Acti detected with IRDye 680LT Goat ati-rabbit. (B) After strippig with stadard strippig Procedure. (C) After strippig for a additioal 20 mi. (D) After additioal 5 mi strippig with the additio of SDS. (E) After 20 miutes total strippig time i NewBlot PVDF + SDS. (F) Reprobe image showig ERK2 detected with IRDye 680LT Goat ati-mouse ad Tubuli detected with IRDye 800CW Goat ati-rabbit.

7 NewBlot PVDF 5X Strippig Buffer Page 7 IV. Frequetly Asked Questios How may times ca a membrae be stripped? Typically, up to three times. The umber of times is depedet o several factors, icludig the type ad amout of boud atige, the type of membrae used, ad the strigecy of strippig coditios. Ca I quatify after strippig ad reprobig? Quatificatio ca be performed to evaluate bad itesities withi the reprobed blot, but ot to compare bad itesities from previous aalyses of the same blot. Oly qualitative aalysis should be performed whe comparig stripped/reprobed blots. Ca I reuse the strippig solutio? The presece of IRDye Ifrared Dye i used strippig solutio will cotribute to high backgroud fluorescece whe reprobig the membrae; therefore, oly the use of freshlyprepared strippig solutio is recommeded. Why do I see high backgroud fluorescece after strippig ad reprobig my membrae? Typically, reblockig the membrae after strippig is ot ecessary. If you see a excessive amout of backgroud sigal, however, additioal blockig may help. Block for miutes i Odyssey Blockig Buffer prior to reprobig your membrae. Why am I still seeig bads eve after strippig for a exteded period of time? Or, why ca I ot see ay bads after reprobig my blot? May factors ca affect overall performace of the NewBlot PVDF Strippig Buffer. Strippig Buffer icubatio time ad temperature ca be icreased, or SDS ca be added to the strippig buffer to ehace the strigecy of the buffer. Please refer to Optimizatio Guide for more iformatio. Ca I use the NewBlot Strippig Buffer for blots probed with alterate dye-labeled atibodies? NewBlot Strippig Buffer has bee used with a umber of fluorescetly-labeled atibodies from other maufacturers, ad works quite well i most cases; however, NewBlot Strippig Buffer is optimized for use with LI-COR IRDye 800CW, IRDye 680LT, or IRDye 680RD secodary atibodies. NewBlot PVDF Strippig Buffer caot be used with IRDye 700DX secodary atibodies. Will NewBlot PVDF Strippig Buffer work with chemilumiescet/colorimetric substrates? NewBlot Strippig Buffer has ot bee tested for use with chemilumiescet detectio or colorimetric substrates; therefore, it is oly recommeded for use with LI-COR fluorescetly labeled secodary atibodies.

8 2014 LI-COR, Ic. LI-COR is a ISO 9001 registered compay. LI-COR, Odyssey, IRDye, ad NewBlot are trademarks or registered trademarks of LI-COR, Ic. i the Uited States ad other coutries. IRDye reagets ad the Odyssey Imagig Systems are covered by U.S. ad foreig patets ad patets pedig. All other trademarks belog to their respective owers. For patet iformatio, visit Superior Street P.O. Box 4000 Licol, Nebraska USA Techical Support: North America: Iteratioal: Fax: LI-COR GmbH Germay, Servig Europe, Africa ad the Middle East: +49 (0) LI-COR UK Ltd. UK, Servig UK, Irelad, Scadiavia: +44 (0) All other coutries, cotact LI-COR Bioscieces or a local distributor: Doc# /14