Whole cell lysates were generated using lamemmli buffer (Bio-rad) in the presence of proteinase inhibitor cocktail. Lysates were run on 10% SDS-PAGE and transferred onto a nitrocellulose membrane. Membranes were cut at approximately 60 kDa; the top portion of the membrane was used to probe for cyclin T1 (CycT1) (75 kDa), the bottom portion was used to probe for CDK9 (40 kDa), Hexim 1 (Hex1) (54 kDa) and βactin (55 kDa). Membranes were blocked in 5% non-fat milk (NFM) for at least 1 hour and blotted overnight with rabbit anti-human cyclin T1 antibody (Santa Cruz Biotechnology), rabbit anti-human CDK9 (Santa Cruz Biotechnology), rabbit anti-human Hex1 (Santa Cruz Biotechnology), and rabbit anti-human β actin (abcam) in 5% NFM. Membranes were washed 3 x in PBS with 0.05% Tween 20, and then blotted for 1 hour with HRP anti-rabbit IgG, in 5% NFM. After washing 3 x with PBS with 0.05% Tween 20; membranes were treated with ECL Plus chemiluminescence reagent (Promega) for 5 minutes and imaged using Odyssey Fc imaging system and Image Studio software (LI-COR). Reprobed membranes were stripped with NewBlot Stripping Buffer (LI-COR) then washed 3 x with PBS.
Newblot stripping buffer
Manufactured by LI COR
NewBlot Stripping Buffer is a laboratory reagent used to remove previously bound antibodies from Western blot membranes, allowing the membranes to be reprobed with new antibodies. The buffer helps to dissociate the antigen-antibody complexes, enabling the reuse of the membrane for multiple analyses.
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2 protocols using newblot stripping buffer
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Western Blot Analysis of Cell Cycle Proteins
2
Optimized Western Blot Protocol
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Whole cell lysates were generated using lamemmli buffer (Bio-rad) in the presence of proteinase inhibitor cocktail. Lysates were run on 10% SDS-PAGE and transferred onto a nitrocellulose membrane. Membranes were cut at approximately 60 kDa; the top portion of the membrane was used to probe for CycT1 (75 kDa) and the bottom portion was used to probe for phospho-(p-) (42 kDa) and total IKBα (40 kDa), p- and total ERK (42 kDa), and β-actin (55 kDa). Membranes were blocked in 5% non-fat milk (NFM) for at least 1 hour and blotted overnight with mouse anti-human cyclin T1 antibody (sc-271348, Santa Cruz Biotechnology), mouse anti-human p-ERK (P-p44/42 MAPK) (9106S, Cell Signaling), rabbit anti-human ERK (p44/42 MAPK) (9102S, Cell Signaling), mouse anti-human p-IKBα (9246S, Cell Signaling), mouse anti-human IKBα (48145S, Cell Signaling) and rabbit anti-human β-actin (ab8227, abcam) antibodies in 5% NFM. Membranes were washed 3 x with PBS with 0.05% Tween 20, and then blotted for 1 hour with HRP anti-rabbit IgG, in 5% NFM. After washing 3 x with PBS with 0.05% Tween 20; membranes were treated with ECL Plus chemiluminescence reagent (Promega) for 5 minutes and imaged using Odyssey Fc imaging system and Image Studio software (LI-COR). Reprobed membranes were stripped with NewBlot Stripping Buffer (LI-COR) then washed 3 x with PBS.
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