---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
date: 2008-11-17 18:32:31+00:00
layout: post
slug: sdspagewestern-purified-his-column-fst-samples-from-20081112
title: SDS/PAGE/Western - Purified (His column) FST samples from 20081112
categories:
  - 2008
  - Miscellaneous
tags:
  - anti-His tag
  - antibody
  - chemiluminescent
  - Coomassie
  - SDS-PAGE
  - SeeBlue Plus
---

10uL of each sample from 20081112 (binding supe, washes and elution fractions) were mixed with 2x sample reducing buffer and boiled 5 mins. Samples were loaded onto Pierce 4-20% Tris-Glycine SDS/PAGE gels. 10uL of SeeBlue ladder was loaded. 5uL of positive control lysate was loaded. Gel was run @ 150V for 45mins.

Gel was transferred to nitrocellulose membrane (equilibrated in tris-glycine transfer buffer for 5mins) @15V for 15mins. Membrane washed briefly in 1x TBS-T, then blocked for 1hr. Gel was stained for 1hr in Coomassie stain and then destained w/ 10% acetic acid until bands were clearly visible.

![](https://eagle.fish.washington.edu/Arabidopsis/SDS-PAGE/20081117.JPG)

Lane #1 - Ladder

Lane #2 - Positive control lysate

Land #3 - Binding supe

Lane #4 - Wash

Lane #5 - Elution fraction #1

Lane #6 - Elution fraction #2

Lane #7 - Elution fraction #3

Lane #8 - Elution fraction #4

Lane #9 - Elution fraction #5

Lane #10 - Elution fraction #6

Lane #11 - Elution fraction #7

Lane #12 - Elution fraction #8

Primary Ab (anti-6x His) was added to membrane at a 1:10,000 dilution. This incubated for 1hr. Used Ab solution was stored @ 4C.

Membrane was washed with 1x TBS-T and fresh blocking solution was added per the [protocol](/protocol#tocWestern).

Secondary Ab (DAR HRP) was added to membrane @ 1:15,000 dilution and incubated for 30mins.

Membrane was washed with 1x TBS-T per the [protocol](/protocol#tocWestern).

Blots were developed using Millipore Immobilon chemiluminescent regent and imaged.



Results:

Western is totally blank. Again. Absolutely no signal at all detected. Not even a smudge/blob/dot/forceps mark/crease. Nothing.
