
What is Western Blotting?
Your common Western blotting questions, answered. Western blotting is a widely used analytical technique that can identify one or more specific proteins in a complex
The six Western blot steps outlined below serve as the complete guide to everything you need for successful Western blot analysis, including equipment, supplies, and reagents.
Keep reading for an in-depth overview of the six Western blotting steps. We’ve also added links to the tools you’ll need, so you can quickly find exactly what’s required to complete your experiments.
For most experiments, the first step to Western blot analysis is separating the proteins in a sample using polyacrylamide gel electrophoresis (PAGE). In SDS-PAGE (where the proteins in the sample are coated with the detergent SDS), proteins migrate according to their size, with smaller proteins migrating more quickly through the gel. The percentage of polyacrylamide in the gel determines how easily proteins of various sizes can move through the gel, with higher percentage gels having a tighter gel matrix better for resolving smaller proteins.
Polyacrylamide gels may be purchased ready-to-use in a variety of percentages or gradients, or gels may be hand cast to achieve a customized percentage.
Once protein separation is complete, the proteins are transferred from the polyacrylamide gel to a solid membrane support. Membranes are usually made from nitrocellulose (NC) or polyvinylidene difluoride (PVDF). Transfer involves assembling a transfer “sandwich” (Figure 1) in which the gel is placed next to the membrane. Both are then placed inside a cassette with blotting paper and sponges on either side to ensure a secure fit within the cartridge. Transfer occurs with the sandwich submerged in transfer buffer in a tank, and a current is passed through the sandwich to drive the proteins from the gel to the membrane.
Before the target protein(s) can be detected on the blot, non-specific binding sites on the membrane must be blocked by incubating the membrane in a blocking buffer. Home-made blocking buffers contain proteins such as dry milk or serum albumin to block non-specific protein-binding sites.
The blocked membrane is incubated with an antibody that binds to the target protein of interest. Incubation conditions depend on the antigen-antibody pair. The primary antibody may be diluted in blocking buffer. Learn more about primary antibodies at Bosterbio. Excess unbound primary antibody is washed away in a series of washes.
The presence of primary antibodies bound to their target protein on the blot is detected by binding a labeled secondary antibody to the primary antibody. Secondary antibodies are usually labeled with a fluorophore that can be detected directly, or bound to an enzyme like horseradish peroxidase (HRP) that reacts with a substrate to produce light (chemiluminescence) or a colored product that can be detected visually using an Azure Imager, another digital imager, or using film.
Finally, the bound secondary antibodies are detected. For chemiluminescent detection, the blot is incubated with a chemiluminescent substrate and the emitted light detected using an Azure 300 Imager, another digital imager, or in a dark room using film. For fluorescent detection, the blot is imaged using an imager such as the Azure 500 that has a light source to excite the fluorophore and the correct filters to detect the emitted fluorescence.
The Azure 500 is a multichannel, multimodal fluorescent imager, with IR, visible light, and UV excitation channels. It allows you to image and quantify two different targets in the same position more efficiently using Near-Infrared (NIR), and normalize to fluorescent total protein stain or a housekeeping protein in the green channel without needing to strip and re-probe your Western blots.
Digital blot images can be analyzed using analysis software, such as AzureSpot Pro. To obtain quantitative information from a Western blot, the signal for the protein of interest can be compared to the signal for a housekeeping protein or to total protein in a process known as total protein normalization (TPN).
Need help with your Western blots and don’t know where to begin?
FREE WESTERN BLOT eBOOK
Related blog posts on Western blotting…
Your common Western blotting questions, answered. Western blotting is a widely used analytical technique that can identify one or more specific proteins in a complex
One of the most common questions when troubleshooting problematic Western blots is, “Why is the background so high?” High or uneven background doesn’t only look bad-
Validating your antibodies is the process of confirming an antibody recognizes your protein of interest with low cross-reactivity to other targets. Validating antibodies is critical
Cookie | Duration | Description |
---|---|---|
cookielawinfo-checbox-analytics | 11 months | This cookie is set by GDPR Cookie Consent plugin. The cookie is used to store the user consent for the cookies in the category "Analytics". |
cookielawinfo-checbox-functional | 11 months | The cookie is set by GDPR cookie consent to record the user consent for the cookies in the category "Functional". |
cookielawinfo-checbox-others | 11 months | This cookie is set by GDPR Cookie Consent plugin. The cookie is used to store the user consent for the cookies in the category "Other. |
cookielawinfo-checkbox-advertisement | 1 year | Set by the GDPR Cookie Consent plugin, this cookie is used to record the user consent for the cookies in the "Advertisement" category . |
cookielawinfo-checkbox-necessary | 11 months | This cookie is set by GDPR Cookie Consent plugin. The cookies is used to store the user consent for the cookies in the category "Necessary". |
cookielawinfo-checkbox-performance | 11 months | This cookie is set by GDPR Cookie Consent plugin. The cookie is used to store the user consent for the cookies in the category "Performance". |
CookieLawInfoConsent | 1 year | Records the default button state of the corresponding category & the status of CCPA. It works only in coordination with the primary cookie. |
elementor | never | This cookie is used by the website's WordPress theme. It allows the website owner to implement or change the website's content in real-time. |
JSESSIONID | session | The JSESSIONID cookie is used by New Relic to store a session identifier so that New Relic can monitor session counts for an application. |
LS_CSRF_TOKEN | session | Cloudflare sets this cookie to track users’ activities across multiple websites. It expires once the browser is closed. |
viewed_cookie_policy | 11 months | The cookie is set by the GDPR Cookie Consent plugin and is used to store whether or not user has consented to the use of cookies. It does not store any personal data. |
woolentor_already_views_count_product | session | Woolentor set this cookie to store product count in cart. |
woolentor_viewed_products_list | 5 days | Woolentor set this cookie to store products in a cart. |
zfccn | session | Zoho sets this cookie for website security when a request is sent to campaigns. |
Cookie | Duration | Description |
---|---|---|
_zcsr_tmp | session | Zoho sets this cookie for the login function on the website. |
S | 1 hour | Used by Yahoo to provide ads, content or analytics. |
Cookie | Duration | Description |
---|---|---|
_uetsid | 1 day | Bing Ads sets this cookie to engage with a user that has previously visited the website. |
_uetvid | 1 year 24 days | Bing Ads sets this cookie to engage with a user that has previously visited the website. |
gaconnector_page_visits | 6 months | This cookie is set to collect information on user behavior and navigation which is used to optimize the website. |
gaconnector_pages_visited_list | 6 months | This cookie is set to collect information on user behavior and navigation which is used to optimize the website. |
SRM_B | 1 year 24 days | Used by Microsoft Advertising as a unique ID for visitors. |
Cookie | Duration | Description |
---|---|---|
_ga | 2 years | The _ga cookie, installed by Google Analytics, calculates visitor, session and campaign data and also keeps track of site usage for the site's analytics report. The cookie stores information anonymously and assigns a randomly generated number to recognize unique visitors. |
_ga_B687YMESCW | 2 years | This cookie is installed by Google Analytics. |
_ga_devsite | 2 years | This cookie is installed by Google Analytics. |
_gat_gtag_UA_43984149_1 | 1 minute | Set by Google to distinguish users. |
_gcl_au | 3 months | Provided by Google Tag Manager to experiment advertisement efficiency of websites using their services. |
_gid | 1 day | Installed by Google Analytics, _gid cookie stores information on how visitors use a website, while also creating an analytics report of the website's performance. Some of the data that are collected include the number of visitors, their source, and the pages they visit anonymously. |
0d269e8747 | session | Zoho PageSense sets this cookie to evaluate visitor, session and campaign data and keeps track of site usage for the site’s analytics report and is set by Zoho Analytics. |
CONSENT | 2 years | YouTube sets this cookie via embedded youtube-videos and registers anonymous statistical data. |
gaconnector_all_traffic_sources | 6 months | This cookie determines how the user accessed the website. This information is used by the website operator to measure the efficiency of their marketing. |
gaconnector_browser | 6 months | This cookie is set to determine the browser. |
gaconnector_city | 6 months | This cookie is set to determine the city. |
gaconnector_country | 6 months | This cookie is set to determine the country. |
gaconnector_country_code | 6 months | This cookie is set to determine the country code. |
gaconnector_device | 6 months | This cookie is set to determine the device. |
gaconnector_fc_campaign | 6 months | This cookie is set to determine the first click campaign. |
gaconnector_fc_channel | 6 months | This cookie is set to determine the first click channel. |
gaconnector_fc_content | 6 months | This cookie is set to determine the first click content. |
gaconnector_fc_landing | 6 months | This cookie is set to determine the first click landing page. |
gaconnector_fc_medium | 6 months | This cookie is set to determine the first click medium. |
gaconnector_fc_referrer | 6 months | This cookie is set to determine the first click referrer. |
gaconnector_fc_source | 6 months | This cookie is set to determine the first click source. |
gaconnector_fc_term | 6 months | This cookie is set to determine the first click term. |
gaconnector_GA_Client_ID | 6 months | This cookie stores the Google Analytics Client ID. |
gaconnector_gclid | 6 months | This cookie is set by GA Connector service to store the Google Click Identifier. |
gaconnector_ip_address | 6 months | This cookie is set to determine the IP address. |
gaconnector_latitude | 6 months | This cookie is set to determine the latitude. |
gaconnector_lc_campaign | 6 months | This cookie is set to determine the last click campaign. |
gaconnector_lc_channel | 6 months | This cookie is set to determine the last click channel. |
gaconnector_lc_content | 6 months | This cookie is set to determine the last click content. |
gaconnector_lc_landing | 6 months | This cookie is set to determine the last click landing page. |
gaconnector_lc_medium | 6 months | This cookie is set to determine the last click medium. |
gaconnector_lc_referrer | 6 months | This cookie is set to determine the last click referrer. |
gaconnector_lc_source | 6 months | This cookie is set to determine the last click source. |
gaconnector_lc_term | 6 months | This cookie is set to determine the last click term. |
gaconnector_longitude | 6 months | This cookie is set to determine the longitude. |
gaconnector_OS | 6 months | This cookie is set to determine the operating system. |
gaconnector_region | 6 months | This cookie is set to determine the region. |
gaconnector_time_passed | 6 months | This cookie stores data on the time spent on the website and its sub-pages, during the current session. |
gaconnector_time_zone | 6 months | This cookie is set to determine the time zone. |
gaconnector2_id | never | This cookie is used by the GA Connector service to gather information regarding visitor activity and related advertising campaigns on the website upon enquiry. |
MR | 7 days | This cookie, set by Bing, is used to collect user information for analytics purposes. |
zabUserId | 1 year | Zoho PageSense sets this cookie to store a unique ID for every user who visits that website. |
Cookie | Duration | Description |
---|---|---|
ANONCHK | 10 minutes | The ANONCHK cookie, set by Bing, is used to store a user's session ID and also verify the clicks from ads on the Bing search engine. The cookie helps in reporting and personalization as well. |
IDE | 1 year 24 days | Google DoubleClick IDE cookies are used to store information about how the user uses the website to present them with relevant ads and according to the user profile. |
MUID | 1 year 24 days | Bing sets this cookie to recognize unique web browsers visiting Microsoft sites. This cookie is used for advertising, site analytics, and other operations. |
NID | 6 months | NID cookie, set by Google, is used for advertising purposes; to limit the number of times the user sees an ad, to mute unwanted ads, and to measure the effectiveness of ads. |
test_cookie | 15 minutes | The test_cookie is set by doubleclick.net and is used to determine if the user's browser supports cookies. |
VISITOR_INFO1_LIVE | 5 months 27 days | A cookie set by YouTube to measure bandwidth that determines whether the user gets the new or old player interface. |
YSC | session | YSC cookie is set by Youtube and is used to track the views of embedded videos on Youtube pages. |
yt-remote-connected-devices | never | YouTube sets this cookie to store the video preferences of the user using embedded YouTube video. |
yt-remote-device-id | never | YouTube sets this cookie to store the video preferences of the user using embedded YouTube video. |
yt.innertube::nextId | never | This cookie, set by YouTube, registers a unique ID to store data on what videos from YouTube the user has seen. |
yt.innertube::requests | never | This cookie, set by YouTube, registers a unique ID to store data on what videos from YouTube the user has seen. |
Cookie | Duration | Description |
---|---|---|
__rafm | session | No description available. |
__rasesh | 2 years 9 months 23 days | No description available. |
_depot_session | session | No description |
_vh_gid | 7976 years 10 months 22 days 20 hours 2 minutes | No description |
_vh_sid | 15 minutes | No description |
_vh_sid_pn | session | No description |
383aeadb58 | session | No description available. |
663a60c55d | session | No description available. |
ac09458e72 | session | No description |
c72887300d | session | No description available. |
COMPASS | 1 hour | No description |
DEVICE_INFO | 5 months 27 days | No description |
e188bc05fe | session | No description available. |
fd6b13af5c | session | No description available. |
gclid | 1 month | No description |
iamcsr | session | No description available. |
ncbi_sid | 1 year | No description available. |
ps_payloadSeqId | 2 hours | No description available. |
SM | session | No description available. |
zabHMBucket | 1 year | No description available. |
zc_consent | 1 year | No description available. |
zc_cu | 1 year | No description available. |
zc_cu_exp | 1 year | No description available. |
zc_loc | session | No description available. |
zc_show | 1 year | No description available. |
zc_tp | 1 year | No description available. |
ZCAMPAIGN_CSRF_TOKEN | session | No description available. |
zft-sdc | 1 hour 2 minutes | No description |
zps-tgr-dts | 1 year | No description |
zpssr* | session | No description |
zsc6339725afa6243a997dc48e2eac3fe89 | 30 minutes | No description |
zsd* | 2 hours | No description |
zsrdbO8X4S | 2 hours | No description |