%PDF-1.5 Protocols 48 Reagent and Materials Preparation Tank Blotting Procedure Prepare the Gel and Membrane Sandwich 49 Assemble the Tank and Program the Power Supply 50 Semi-Dry Blotting Procedure51 Trans-Blot® Turbo™ Blotting Procedure 52 Microfiltration 53 Blot Stripping and Reprobing 54 Total Protein Detection 55 %PDF-1.5 %���� f����L�ˁ�@��L= q�N�?��/� k�) It uses specific antibodies in order to identify these proteins, often from a mixture of proteins. Posted in Antibodies, Assay Kits, Biology Cells, cDNA, ... Vector & Virus, Western Blot. stream The guide is intended to be an educational resource to introduce the method rather than a benchtop protocol… 2. h޴RMo�0�+. 0 western blotting protocol. The following guide discusses the entire process of producing a Western blot: sample preparation, gel electrophoresis, transfer from gel to membrane, and immunostain of the blot. 2. ��Ќ��HNK;M�P�(3{�pP'���C���(�D2lB1�:�-�g��ed��7O���Q�I��\�S%���0w��3*N�!0�q+Ff����J�Q�7�`����*M�VMjI��9V� �]� ����!T�-T���-T�Z�(Cn���֐��J�,���%�O��Px�$��:���V|�Gf��3,�X�~(Ɵ�[email protected]�&m2ɏr6 Z��Ȑ���%��=ur INTRODUCTION The western blot (sometimes called the protein immunoblot) is a widely accepted analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. Load equal amounts of protein (20 μg) into the wells of a mini (8.6 x 6.7 cm) or midi (13.3 x 8.7 cm) format SDS-PAGE gel, along with molecular weight markers. Western Blot Protocol SECTION 1 – Equipments & Reagents 1.1 Equipment(s) Shaker (TKB OS701) AutoChemi System (UVP) 1.2 Blocking buffer (also Dilution buffer) Weigh non-fat milk 5 g and dissolve in 100 mL 1X PBST (0.2%) to a final mixture of 5% non-fat milk/PBST (0.2%). 4 0 obj Western blotting uses antibodies to identify individual proteins within a cell or tissue lysate. 1 0 obj 533 0 obj <>stream Set on Electrode Plate See the troubleshooting section for guidance. If the positive control lysate does not result in a positive signal, the western blotting protocol requires optimization. i�L������a�f ��&�6�3#5��T `%�P�kA�X�� �~��=��2�_�{���Jک�0jt�5���')Kn�(y)��cఀ��0� �P�WZ��in��%��������$h��?�}�q�a�R�RKHV�a�;i�f�L��%�/�02v�8o����aD��ߔ*�y��(h������ɖ�U����d���L�{�: �vTq}F�Y���6�A~\X��(h�>��W�A�}�4���Z[(���|����Z�&. Place the cell culture dish on ice and wash the cells with ice-cold PBS. 3. See the troubleshooting section for guidance. <>/ExtGState<>/XObject<>/ProcSet[/PDF/Text/ImageB/ImageC/ImageI] >>/MediaBox[ 0 0 595.32 841.92] /Contents 4 0 R/Group<>/Tabs/S/StructParents 0>> `j�&� @^[email protected]��!�AY���U�����[���[�W��Gr8/r����~s���������~��ȇ��������a�n}����v\]|��G����j�={F^�zI���:���F:����+u����'d��^\�g��q�iry=�!�#�0)����q�[email protected];�0���,� ��j�����k��������H.��^Â���60rI`k� 5�E9�����/�x�8���7w���W[�WWځ^3��.���:b`"��]%T8>�Ĩ���O��τ�̸$� 3. 2-1. 3 0 obj Blots can be imaged immediately while still wet, or alternatively may be dried prior to imaging. 2 0 obj Alexa Portugal Audrey Peng MCB 253 15 October 2020 General Western Blot Protocol Background: The purpose of the General Western Blot Protocol is to identify proteins that have been separated based on their molecular molecular weight by gel electrophoresis. ���,19��KBb� Շ,�v~�j2/��+�B�FZQ��N��K}�w�N5~��՘�[|�Q,BCZ�W3�p��A���^�!1���U�aUy�(RV�\`��� R�^�����k2��[email protected]\�d�x�f�-+��\�, ��(|���k��f���owb�ְ��,��X4h��V��5Y�WHkB�4gY3�I��M�@�H�-5�/d��ls�h�P��y��b�М��@���� ���b�d��P�v0%X�SR�p!\�)@>��X1��[email protected]&��2��z�A���(����bwtP+�0002ݸd��.8ɚ �������t�Y�.1�k��ta�bp}�nt���5� ^� ֊�� �X+�Jx,X�Mu��`ae�{���"�!���Rl`y�џA���{�T���+�Q � �)r <> Western blots are used to determine the identity, size, and abundance of specific proteins within a sample. 30650-31) into another clean tray, hereafter referred to as Tray 2. x��YKo7���Q <>>> h�b```f``�d`211 �+P����@�E�)YıY`�d�f �f��:M S��/Ɲ��/�z�����E�՗�8��9��7��3Ml�x�}!$�oR��{ ��I6�N�^i�ɱ�13��Ap�ӥ��Ӟ��X4�����iO6]���E���% �At�g���� 9�&]��\��4]�; Drying the membrane allows for extended storage of the blot and can reduce exposure times. endobj A western blot experiment, or western blotting, is a routine technique for protein analysis.