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Anti-Beta-2-microglobulin [BBM.1] in Immunocytochemistry (HeLa stable cells)

Addgene #207115

Data Submitted By
Meghan Rego and Amrita Rhoads
Lab Name
Addgene
Submission Date
October 09, 2024
Publication Date
October 18, 2024 (modified November 21, 2024)
Abstract
HeLa cells stably expressing a scrambled shRNA or shRNAs directed against B2M were probed with Anti-Beta-2-microglobulin [BBM.1] (Addgene #207115) recombinant monoclonal antibody. B2M signal was reduced in the cells expressing shB2M.
HeLa cells were transduced with shRNA expressing lentiviral particles and stable cell lines selected and expanded. Stable cell lines expressing shScrambled have a strong cytoplasmic B2M signal. The signal is reduced in the lines expressing shRNAs against B2M (shB2M_255 and shB2M_256). Nuclei were counterstained with DAPI.
Caption: HeLa cells were transduced with shRNA expressing lentiviral particles and stable cell lines selected and expanded. Stable cell lines expressing shScrambled have a strong cytoplasmic B2M signal. The signal is reduced in the lines expressing shRNAs against B2M (shB2M_255 and shB2M_256). Nuclei were counterstained with DAPI.
Image attribution: Amrita Rhoads and Meghan Rego, Addgene

This report is made available under the Creative Commons Attribution 4.0 International License.

Antibodies Used
Anti-Beta-2-microglobulin [BBM.1]
Addgene #207115
Goat anti-mouse IgG2a cross-adsorbed secondary antibody
Thermo Fisher #A21131

Materials & Methods

Sample

Species
Human
Cell / Tissue Type
HeLa
Additional Information
HeLa cells stably expressing shRNA from lentivirus.

Buffer

Name
PBS
pH
7.4

Fixative

Fixative
Fixed and permeabilized with 3% paraformaldehyde powder and 0.1% Triton X-100 in PBS/Ca/Mg
Time
30 min
Temperature
4 ˚C

Blocking

Agent
Milk
Concentration
4% w/v
Time
45 min
Temperature
Room Temperature

Primary Antibody

Name
Anti-Beta-2-microglobulin [BBM.1]
Source
Addgene
Catalog Number
207115
Species
Mouse
Gene / Target
B2M
Concentration
0.2 µg/mL
Time
45 min
Temperature
Room Temperature
View in Addgene Catalog

Secondary Antibody

Name
Goat anti-mouse IgG2a cross-adsorbed secondary antibody
Source
Thermo Fisher
Catalog Number
A21131
Concentration
1:2500
Conjugate
Alexa Fluor 488
Time
45 min
Temperature
Room Temperature

Additional Information

Additional Information
shRNAs obtained from Millipore Sigma (IDs: TRCN0000057255 and TRCN0000057256)
Protocol
Trimmer lab immunocytochemistry protocol
Controls
HeLa cells expressing a scrambled shRNA showed expected labeling and the labeling was absent or reduced in the shB2M cells.

Results

Result
Pass (The antibody worked under these conditions)
Describe Results
HeLa cells expressing scrambled shRNA have strong cytoplasmic B2M labeling. The labeling was reduced in the shB2M_255 cell line and undetectable in the shB2M_256 cell line.