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TBS 10X alternative recipe (concentrated Tris-buffered saline) For 1 L 24 g Tris-HCl (formula weight 157.6 g) 5.6 g Tris base (formula weight 121.1 g) 88 g NaCl (formula weight 58.4 g) Dissolve in 900 mL distilled water 1. 0000004897 00000 n
Prepare a 100 mM sodium orthovanadate solution with double distilled water. Western Blot washing buffer: 1X PBST (Phosphate-buffered saline, 0.1% Tween 20) For 1.0 L: 100 mL 10X PBS 899 mL ddH 2 O 1 mL Tween 20: 1X TBST (Tris-buffered saline, 0.1% Tween 20) For 1.0 L: 100 mL 10X TBS 899 mL ddH 2 O 1 mL Tween 20: Blocking buffer: Western Blot blocking buffer: 5% (or 3%) nonfat dried milk or BSA in 1X TBST or 1X PBST. ��0E�SX�Ա���Ԇ#
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Wash the membrane 3 times with agitation for 10 minutes each in wash buffer. 116 0 obj
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Ensure the volume of the antibody solution is enough to fully cover the membrane. -��*Uu �,��d�[�&�q܋�����n��#l��.���~�����?ƾ>�ɝۮ���N��vYY����Go~����i��~��uߝ�l�t����6�w��n�S|���cǶ�����ó����7^c7�V���T��q�v�F���^�Mz�N��4��_�!�j��&ކc�cw�ٲH��-�ҵ�b������J~/�_��k���;0�LMbΗ�l�9��\�$���\�=ك�,`�y�y%����t�p�t�p�t�p����:����A� p:����A� p:dCހ�� 7an�܄� rz 0000014467 00000 n
Set pH to 9.0 with HCl. Incubate the membrane protein-side up in the primary antibody solution with agitation, for 1 hour at room temperature or overnight at 2–8°C. Sometimes, ponceau red staining is an alternative to check whether the protein transfer is successful, so a recipe of ponceau red staining solution is necessary. 0000004243 00000 n
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Directions for 1X Transfer Buffer: 1) Dissolve Tris base and glycine together in 1.6 L of ddH2O. 0000030124 00000 n
2) Add methanol and mix. 0000001381 00000 n
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Besides, TBS buffer, blocking buffer, and TBST buffer (washing buffer) are also needed to be prepared. }���9�|>��k�y;ݞ���nCrż_��t�:�UwJY��k��7�VY�����ñ~�Ƭ\~��U�_V��ګ�ż��t������/�8_l�7�[�-4}l1M[�ҳ�G}��^B�����B-�����J
f�#49=8=9=8��zmZ�+�� To denature, use a loading buffer with the anionic detergent sodium dodecyl sulfate (SDS), and boil the mixture at 95–100°C for 5 min. 0000011772 00000 n
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Tris-glycine native running buffer: 25 mM Tris base, 192 mM glycine, pH 8.3 Recipe for 10X buffer stock: Tris base 29 g Glycine 144 g Deionized water to 1,000 mL MOPS SDS running buffer: 50 mM MOPS, 50 mM Tris base, 0.1% SDS, 1 mM EDTA, pH 7.7 Recipe for 20X buffer stock: MOPS 104.6 g Tris base 60.6 g SDS 10 g EDTA 3.0 g Deionized water to 500 mL 0000008733 00000 n
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Dilute the primary antibody in 15 ml of 5% non-fat dry milk in TBST. 0000002540 00000 n
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Cool to room temperature. 116 33
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H�tVMr55ܿS�b,[����8B Set pH to 9.0 … |��_���W�+�z ��^/���K�AO�=�D�AO�=�$�'= � � � � � � ��=�'�'�'�G�Q�QYSQSYSQSYSQS�Q�Q��M@���������w�������ٕ�����!�9d���=�3�3�3�3�3�3�3�3�3�3�3�3�3�3���} 0000000016 00000 n
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Loading buffer, running buffer, coomassie brilliant blue staining solution, and coomassie destaining solution are needed to be prepared for SDS-PAGE, while western blot transfer buffer (recipe here is for wet transfer) preparation is required for protein transfer. 0000030420 00000 n
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